U.S. patent application number 14/129773 was filed with the patent office on 2014-12-18 for process and apparatus for quantifying nucleic acid in a sample.
This patent application is currently assigned to BIOQUANTA SA. The applicant listed for this patent is Marc Conti, Nicolas Delacotte, Carlosse Keumeugni Kwemo, Sylvain Loric, Philippe Manivet, Mamadou Saliou Bah. Invention is credited to Marc Conti, Nicolas Delacotte, Carlosse Keumeugni Kwemo, Sylvain Loric, Philippe Manivet, Mamadou Saliou Bah.
Application Number | 20140371082 14/129773 |
Document ID | / |
Family ID | 47424831 |
Filed Date | 2014-12-18 |
United States Patent
Application |
20140371082 |
Kind Code |
A1 |
Conti; Marc ; et
al. |
December 18, 2014 |
PROCESS AND APPARATUS FOR QUANTIFYING NUCLEIC ACID IN A SAMPLE
Abstract
In one aspect, the present invention concerns methods and kits
for quantifying a target nucleic acid in a sample. In embodiments,
a method comprises sequestrating the target nucleic acid by one or
more interactors to form a sequestered conjugate that will be
further labeled to form a labeled conjugate. The signal produced by
the labeled conjugate is measured and correlated to the amount of
the target nucleic acid. In another aspect, the present invention
concerns a method for quantifying a target nucleic acid in a sample
by a reaction of the nucleic acids in the sample with a nucleic
acid interactor or set of interactors to form a conjugate. The
conjugate is then sequestered from the rest of the sample with a
molecule bound to a support, to form a sequestered conjugate. The
sequestered conjugate is labeled to form a labeled conjugate. The
signal produced by the labeled conjugate is measured and correlated
to the amount of the target nucleic acid. In some embodiments, the
target nucleic acid is cell free nucleic acids. In embodiments, the
interactor binds to nucleic acids in the sample in a nonsequence
specific manner.
Inventors: |
Conti; Marc; (Palaiseau,
FR) ; Loric; Sylvain; (Brunoy, FR) ; Manivet;
Philippe; (Paris, FR) ; Delacotte; Nicolas;
(L'Hay-les Roses, FR) ; Keumeugni Kwemo; Carlosse;
(Villeneuve La Garenne, FR) ; Saliou Bah; Mamadou;
(Evry, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Conti; Marc
Loric; Sylvain
Manivet; Philippe
Delacotte; Nicolas
Keumeugni Kwemo; Carlosse
Saliou Bah; Mamadou |
Palaiseau
Brunoy
Paris
L'Hay-les Roses
Villeneuve La Garenne
Evry |
|
FR
FR
FR
FR
FR
FR |
|
|
Assignee: |
BIOQUANTA SA
Paris
CO
ASSISTANCE PUBLIQUE-HOPITAUX DE PARIS
Paris
BIOQUANTA CORP.
Aurora
|
Family ID: |
47424831 |
Appl. No.: |
14/129773 |
Filed: |
July 2, 2012 |
PCT Filed: |
July 2, 2012 |
PCT NO: |
PCT/US2012/045271 |
371 Date: |
July 31, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61503599 |
Jun 30, 2011 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/6.1;
436/501; 506/16 |
Current CPC
Class: |
C12Q 1/68 20130101; C12Q
2522/101 20130101; C12Q 2537/125 20130101; C12Q 2537/125 20130101;
C12Q 2537/161 20130101; C12Q 2545/113 20130101; C12Q 1/68 20130101;
C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12Q 2563/173 20130101;
C12Q 2537/125 20130101; C12Q 2545/114 20130101; C12Q 2563/173
20130101; C12Q 2545/113 20130101; C12Q 1/6806 20130101 |
Class at
Publication: |
506/9 ; 436/501;
435/6.1; 506/16 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for quantifying a cell free target nucleic acid in a
sample comprising the steps of: (a) contacting the cell free target
nucleic acid in the sample with at least one non sequence specific
nucleic acid interactor to form a conjugate; (b) sequestering the
conjugate by reacting the conjugate with a capture molecule bound
to a support; (c) labeling the sequestered conjugate with a signal
generating molecule to form a labeled conjugate; (d) measuring
signal of the labeled conjugate; wherein the signal of the labeled
conjugate is indicative of the amount of the target nucleic acid in
the sample.
2. A method for quantifying a cell free target nucleic acid in a
sample comprising the steps of: (a) providing a known quantity of
nucleic acid labeled with a signal generating molecule into the
sample containing the target nucleic acid; (b) reacting the cell
free target nucleic acid and the labeled nucleic acid with a
sequestered non sequence specific nucleic acid interactor to form a
sequestered target conjugate and a sequestered labeled conjugate,
wherein the target nucleic acid competes with the labeled nucleic
acid for binding to said sequestered interactor; (c) measuring the
signal of the sequestered labeled conjugate; and (d) determining
the amount of said target nucleic acid by comparing the signal in
step (c) with that detected by sequestered labeled conjugate in the
absence of target nucleic acid.
3. A method for quantifying a cell free target nucleic acid in a
sample comprising the steps of: (a) contacting the target nucleic
acid in the sample with a sequestered non sequence specific nucleic
acid interactor to form a sequestered conjugate; (b) contacting the
sequestered conjugate with a second non sequence specific nucleic
acid interactor labeled with a signal generating element to form a
labeled conjugate; and (c) measuring signal of the labeled
conjugate; wherein the signal of the labeled conjugate is
indicative of the amount of the target nucleic acid in the
sample.
4. The method of claim 1, wherein steps (a) and (b) are carried out
in the same vessel.
5. The method of claim 1 wherein said nonsequence specific nucleic
acid interactor is cell impermeant.
6. The method of claim 1 wherein the nucleic acid interactor is a
set of at least two nucleic acid interactors.
7. The method of claim 1 wherein the nucleic acid interactor is an
intercalating agent.
8. The method of claim 1 wherein the sample comprises molecules
other than nucleic acids.
9. The method of claim 1 wherein the nucleic acid interactor is a
nonspecific single stranded nucleic acid.
10. The method of claim 1 wherein the nucleic acid has not been
isolated or purified.
11. The method of claim 2, wherein the molecule bound to the
support is a nucleic acid intercalator.
12. The method of claim 3, wherein steps (a), (b) and (c) are
carried out in the same vessel.
13. The method of claim 11, further comprising reacting the
conjugate with a nucleic acid intercalating agent prior to
attaching the sequestered conjugate with a signal generating
molecule.
14. The method of claim 1 wherein the interactor is selected from
the group consisting of acridine, picogreen, pop03, yo yo 1, and
combinations thereof.
15. The method of claim 1, wherein the target nucleic acid is
double-stranded nucleic acid.
16. A kit for quantifying a target nucleic acid in a sample
comprising (i) a first nucleic acid interactor (ii) optionally, a
second nucleic acid interactor; (iii) a label, optionally bound to
the first interactor; (iv) instructions for use.
17. The kit of claim 16 further comprising control nucleic acid
fragments of different sizes and sequences.
18. A kit for quantifying a target nucleic acid in a sample
comprising (i) a first nucleic acid interactor, wherein the first
nucleic acid interactor is non sequence specific and comprises a
moiety that allows the first nucleic acid interactor to be
sequestered; (ii) a second nucleic acid interactor that is
different from the first nucleic acid interactor; (iii) a label,
optionally bound to the second interactor; (iv) instructions for
use.
19. A kit of claim 18, wherein the first nucleic acid interactor is
a nucleic acid intercalator.
20. A kit of claim 18, wherein the first nucleic acid interactor is
linked to a solid substrate though a linker.
Description
FIELD OF THE INVENTION
[0001] The invention relates to processes that ensure an easy
measurement of even a small quantity of nucleic acids, without the
need for sample extraction, purification, or amplification. The
invention is adapted to various kinds of readout methods. The
processes are suitable for manual handling, semi-automation or
automation. The present processes are suitable for a fully
automated operation, and can be carried out in one or several
vessels, containers or wells, without further handling of the
samples or the reaction components or the entire reaction mixture,
with only an eventual transfer of reacting material by automated
means from one vessel to another.
[0002] The invention further relates to a method for quantifying
cell-free nucleic acids, i.e., nucleic acids present outside cells.
It finds use, inter alia, in ascertaining the existence of an
abnormality characterized by the incidence of cell free nucleic
acid in the bloodstream of a patient, and in monitoring a patient
previously presenting with such an abnormality and after such
patient has been treated to detect possible recurrence of the
abnormality.
BACKGROUND OF THE INVENTION
[0003] After cellular disruption, such as apoptosis, nuclear DNA
spills into the extracellular environment and is carried by the
blood. This DNA is referred to as cell-free DNA (or free DNA) and
its existence has been known for many years (Mandel & Metais,
1948; Tan et al., 1966; Koffer et al., 1973). Healthy individuals
typically have about 500-1000 genome equivalents of free DNA per
milliliter of blood. A value in this range corresponds to a normal
rate of cellular disruption. A higher level may be an indication of
a disorder that clinicians have to explore and treat. Accordingly,
accurate, fast, relatively low-cost and convenient measurement of
cell-free DNA is desirable.
[0004] To date, high concentrations of cell-free DNA have been
correlated with various pathologies (e.g., lung cancer, ovarian
cancer, breast cancer, melanoma, adrenal hyperplasia, cervical
cancer, preeclampsia, fetal Down's syndrome, nasopharyngeal
carcinoma, leukemia, gastrointestinal malignancy, lupus
erythematosus, rheumatoid arthritis); consequently, cell-free DNA
is beginning to be used as a marker for these diseases.
[0005] In addition, the quantification of cell-free DNA in plasma
can be used to predict the likelihood of serious cardiovascular
complications, which permits appropriate therapy to be given at an
early stage in an attempt to lower the incidence of such
complications.
[0006] The detection and quantification of cell-free DNA has also
added a new dimension to medical diagnosis. In oncology, various
tumor-associated molecular alterations have been detected in the
plasma/serum of cancer patients. In pregnant subjects, the
discovery of fetal DNA in maternal circulating blood has provided a
new source of fetal genetic material for the noninvasive analysis
of numerous fetal conditions, and for the detection of particular
pregnancy-associated disorders. The measurement of cell-free DNA
has also found potential applications in the post-treatment
monitoring of transplant patients and in the assessment and
prognosis of trauma patients.
[0007] Radioimmunoassay techniques have historically been employed
to detect high concentrations of cell free DNA in patients with
cancer, as compared to healthy subjects, or subjects with benign
tumors (Leon et al., 1977; Shapiro et al., 1983). Numerous studies
have reported that high concentrations of cell free DNA can serve
as a diagnostic marker or of progression of breast or lung cancer,
or could be useful in the follow-up of patients treated by
chemotherapy.
[0008] In addition, sub-physiological concentration of cell free
DNA may signify an altered pathology, which may require medical
treatment adjustment.
[0009] Despite the fact that non-physiological concentrations of
cell-free DNA were known to be correlated with various disease
states, including cancer, cell-free DNA has not been used as a
biological marker until the advent of quantitative PCR (qPCR). A
main reason for this is that radioactive assays, although able to
detect cell free DNA, were not optimal due to accompanying safety
concerns and lack of both sensitivity and convenience of
implementation.
[0010] Sensitive, non-invasive techniques such as qPCR allow the
clinician to avoid, or limit, more invasive tests like tissue
biopsies for the detection of cancer. Assaying cell free DNA
content by qPCR can also serve as a screen, at an early stage of
lung, breast or prostate cancer. Moreover, the measurement of
cell-free DNA content by qPCR can be used as a complementary
technique to that of classical marker detection (e.g., by cell
sorting methodologies) in the follow-up of post-operative patients,
patients with pathology (Chang et al., 2003), or patients diagnosed
or treated for cancer (Lam et al., 2003).
[0011] Although qPCR is extremely sensitive in terms of detection
limit, it has several practical and technical drawbacks. Careful
consideration has to be paid to reaction set-up, including reagent
concentrations, contamination from external sources, and purity of
sample. False positives can arise if the reaction components and
conditions are not diligently controlled. Crude samples usually
need to be purified before their use in a qPCR assay to remove
non-nucleic acid components of the sample which inhibit the
amplification reaction or quench the reporter molecule's
fluorescence. Since the amplification procedure is exponential,
small differences in reaction component concentration (e.g.,
polymerase, fluorescent probe, salt, etc.) that may exist from
reaction-to-reaction, can lead to large differences in the amount
of template detected, even while keeping the template concentration
constant.
[0012] The majority of nucleic acid quantification methods (e.g.,
radioimmunological methods, qPCR, and other fluorometric detection
methods, such as ELISA) require the use of purified nucleic acid
samples as templates for the quantification assays. In the case of
ELISA, antibodies can bind to non-nucleic acid components of the
sample. In the case of qPCR, Taq polymerase activity can be
inhibited by components of the crude sample.
[0013] Using the methods described above, it typically takes at
least 3 hours for the entire process of DNA isolation/purification
and quantification to be completed. The time period necessary for
nucleic acid quantification by current methodologies limits these
tools for use in point-of-care diagnostics and on-site forensic
applications.
[0014] Work on cell-free nucleic acids started in 1948 but the last
ten years haven given rise to 80% of the publications on the
subject, reflecting a growing interest and need in this diagnostic
marker. There is a particular need for a rapid automated process to
measure cell-free DNA that could be performed on the available
machines used in clinical diagnostic services. And this need is not
fulfilled with the qPCR methods developed.
[0015] A viable alternative for the detection of DNA in crude
samples is the use of a luminescent readout. Superior sensitivity,
wide dynamic range, low instrument cost and low background are all
characteristics of luminescent methods. For example, luminometry is
up to 100,000 times more sensitive than absorption spectroscopy and
is at least 1,000 times more sensitive than fluorometry. The
unmatched sensitivity of luminescent reactions allows
quantification of nucleic acids without amplification steps.
[0016] In addition, safety, ease of handling and significantly
lower disposal costs are advantages of luminescent assays over
analogous radiography methods.
[0017] Luminescent assays require simpler instrumentation as
compared to fluorescence assays because there is no need for a
light excitation source. Luminescence can be quantified since light
output is directly proportional to the number of luminescent
substrates in the reaction (when volume is held constant from
reaction-to-reaction). Undesirable autofluorescence, background
emission and scatter are eliminated with the use of luminescence
because no excitation light is necessary to generate a signal. In
luminescent reactions, temperature and extraneous light must be
controlled for since both affect reaction rates.
[0018] Although luminescent assays afford the advantages listed
above, they have not been coupled to DNA purification and
subsequent quantification in one integrated assay. A main reason
for this is the inability to accurately quantify luminescence in an
environment containing components other than the target that is to
be quantified. The present invention sets out to solve this problem
with an integrated purification/quantification assay.
[0019] If the luminescence is an easy way to quantify cell-free
nucleic acids, it is possible to envisage other readout methods,
once nucleic acid is captured from the sample by an interactor or
preferably by two or more interactors. So we propose to integrate
in the same general process, capture, extraction and measurement of
cell-free nucleic acids. Capture and extraction will be done by the
interactor, and measure of the signal emitted by a label excited or
otherwise revealed at the end of the process. The signal has to be
sensitive enough to ensure measurement of small amounts of cell
free nucleic acids. It may be, without limitations, optic (UV,
Visible, IR absorption), fluorescent, luminescent or magnetic.
SUMMARY OF THE INVENTION
[0020] The present invention serves as an alternative to
amplification-based nucleic acid detection methods which require
prior nucleic acid purification/isolation. The present invention
also integrates the processes of purification and detection of
nucleic acids. In some embodiments, the present disclosure provides
detection of cell free nucleic acids in a sample without
amplification and/or isolation of nucleic acids in the sample.
[0021] The present invention is suitable for manual handling or
semi-automated or automated processes.
[0022] In case of automation, processes can be carried out during a
unique fully automated process, in one or several vessels,
containers or wells, with automatic transfer of reacting material
by the machine or another machine.
[0023] In one aspect, the present invention integrates capture,
labeling, detection and quantification of nucleic acids in a
sample, and can be carried out in one automated process and in a
single vessel.
[0024] The present invention can be carried out with various
existing or novel automatic analyzers.
[0025] Our data indicated that the interaction between cell-free
nucleic acids and the interactor was dependent on both the sequence
and length of the nucleic acids. Thus, the use of one kind of
interactor is not enough to be sure to measure all cell-free
nucleic acids contained in a sample. A set of two (or more)
interactors, such as three or four, etc., is preferably utilized in
the methods described herein. An integrated procedure that both
isolates and quantifies nucleic acids from a crude sample, with the
use of various readout methods, is disclosed herein. The methods
provided, as compared to nucleic acid purification/detection
schemes previously developed (e.g., DNA isolation by chromatography
followed by qPCR), are compatible with automation, reduce reagent
cost, overall reaction set-up time, as well as time for detection,
because nucleic acids are not amplified, or transferred to a second
reaction zone after isolation.
[0026] In one aspect, the present invention concerns a method for
quantifying a target nucleic acid in a sample by sequestration of
the target nucleic acid by one or more interactors to form a
sequestered conjugate that will be further labeled to form a
labeled conjugate. The signal produced by the labeled conjugate is
measured and correlated to the amount of the target nucleic acid.
In another aspect, the present invention concerns a method for
quantifying a target nucleic acid in a sample by a reaction of the
nucleic acids in the sample with a nucleic acid interactor or set
of interactors to form a conjugate. The conjugate is then
sequestered from the rest of the sample with a molecule bound to a
support, to form a sequestered conjugate. The sequestered conjugate
is labeled to form a labeled conjugate. The signal produced by the
labeled conjugate is measured and correlated to the amount of the
target nucleic acid. In some embodiments, the target nucleic acid
is cell free nucleic acids. In embodiments, the interactor binds to
nucleic acids in the sample in a nonsequence specific manner.
[0027] In yet another aspect, the present invention concerns a
method for quantifying a target nucleic acid in a sample
introducing a known quantity of a nucleic acid labeled with a
readable molecule into the sample containing the target nucleic
acid. The target nucleic acid and the labeled nucleic acid are
sequestered by a sequestered nucleic acid interactor (or set of
interactors) to form a sequestered target conjugate and a
sequestered labeled conjugate wherein the target competes with the
labeled nucleic acid for binding to the interactor. The signal of
the labeled conjugate is then measured. The amount of target
nucleic acid is then derived by comparing the signal of the labeled
conjugate in the presence of target nucleic acid with that emitted
by sequestered labeled conjugate in the absence of target nucleic
acid.
[0028] In some embodiments, a method for quantifying a target
nucleic acid in a sample comprises: (a) contacting the target
nucleic acid in the sample with a sequestered nucleic acid
interactor to form a sequestered conjugate; (b) adding at least one
more nucleic acid interactor; (c) labeling the sequestered
conjugate with a signal generating molecule to form a labeled
conjugate; and (d) measuring the signal of the labeled conjugate;
wherein the signal of the labeled conjugate is indicative of the
amount of the target nucleic acid in the sample. In embodiments,
the method further comprises comparing the amount of the target
nucleic acids to a control sample to determine if the amount of
target nucleic acids of the sample is indicative of disease. In
embodiments, a method further comprises communicating the result to
a health care provider so that the health care provider can conduct
additional diagnostic tests. In some embodiments, the target
nucleic acid is cell free nucleic acids. In embodiments, the
interactor binds to nucleic acids in the sample in a nonsequence
specific manner.
[0029] In other embodiments, a method for quantifying a target
nucleic acid in a sample comprises: (a) contacting the nucleic acid
in the sample with at least one nucleic acid interactor to form a
conjugate; (b) sequestering the conjugate by reacting the conjugate
with a capture molecule bound to a support; (c) labeling the
sequestered conjugate with a signal generating molecule to form a
labeled conjugate; (d) measuring signal of the labeled conjugate;
wherein the signal of the labeled conjugate is indicative of the
amount of the target nucleic acid in the sample. In some
embodiments, the method further comprises comparing the number or
amount of the target nucleic acids to a control sample to determine
if the number or amount of target nucleic acids of the sample is
indicative of disease or disorder. In some embodiments, a method
further comprises communicating the result to a health care
provider so that the health care provider can conduct additional
diagnostic tests. In some embodiments, the target nucleic acid is
cell free nucleic acids. In embodiments, the interactor binds to
nucleic acids in the sample in a nonsequence specific manner.
[0030] In yet other embodiments, a method for quantifying a target
nucleic acid in a sample comprises: (a) providing a known quantity
of nucleic acid labeled with a signal generating molecule into the
sample containing the target nucleic acid; (b) reacting the target
nucleic acid and the labeled nucleic acid with a sequestered
nucleic acid interactor to form a sequestered target conjugate and
a sequestered labeled conjugate, wherein the target nucleic acid
competes with the labeled nucleic acid for binding to said
sequestered interactor; (c) measuring the signal of the sequestered
labeled conjugate; and (d) determining the amount of said target
nucleic acid by comparing the signal in step (c) with that detected
by sequestered labeled conjugate in the absence of target nucleic
acid. In embodiments, the method further comprises comparing the
amount of the target nucleic acids to a control sample to determine
if the amount of target nucleic acids of the sample is indicative
of disease or disorder. In embodiments, a method further comprises
communicating the result to a health care provider so that the
health care provider can conduct additional diagnostic tests. In
some embodiments, the target nucleic acid is cell free nucleic
acids. In embodiments, the interactor binds to nucleic acids in the
sample in a nonsequence specific manner.
[0031] In a specific embodiment, a method for quantifying a target
nucleic acid in a sample comprises: contacting the target nucleic
acid in the sample with a sequestered nucleic acid interactor to
form a sequestered conjugate; contacting the sequestered conjugate
with a second nucleic acid interactor labeled with a signal
generating element to form a labeled conjugate; and measuring
signal of the labeled conjugate; wherein the signal of the labeled
conjugate is indicative of the amount of the target nucleic acid in
the sample. In some embodiments, the method further comprises
comparing the amount of the target nucleic acids to a control
sample to determine if the amount of target nucleic acids of the
sample is indicative of disease or disorder. In some embodiments, a
method further comprises communicating the result to a health care
provider so that the health care provider can conduct additional
diagnostic tests. In some embodiments, the target nucleic acid is
cell free nucleic acids. In embodiments, the interactor binds to
nucleic acids in the sample in a nonsequence specific manner.
[0032] Another aspect of the disclosure provides a kit for
quantifying a target nucleic acid in a sample comprising (i) a
first nucleic acid interactor; (ii) optionally, a second nucleic
acid interactor; (iii) a label, optionally bound to the first
interactor; and (iv) instructions for use.
[0033] Since the interaction between cell-free nucleic acids and a
single interactor was dependent on both the sequence and length of
the nucleic acids, the use of one kind of interactor may not be
enough to ensure the measurement of all cell-free nucleic acids
contained in the medium. So the invention preferably uses two or
more interactors to improve the accuracy of the process.
[0034] In one aspect, the nucleic acid to be quantified in the
sample is cell-free DNA.
[0035] The foregoing methods are useful, inter alia, in
ascertaining the existence of an abnormality characterized by the
incidence of cell free nucleic acid in the bloodstream of a
patient, and in monitoring a patient previously presenting with
such an abnormality (after such patient has been subjected to
therapy to abate or eliminate the abnormality) to detect possible
recurrence of the abnormality. When these methods are employed,
upon the finding of an abnormality, further tests can be employed,
such as DNA assays known in the art, to determine the nature of the
abnormality. Thus, embodiments of the invention contemplate
performance of the methods disclosed herein in tandem with sequence
specific DNA detection methods.
DRAWINGS
[0036] FIG. 1A is a general diagram of a sandwich assay embodiment
of the invention, showing target nucleic acid free in solution
(top) and sequestered (bottom).
[0037] FIG. 1B is a general diagram of a competitive assay
embodiment of the invention showing target nucleic acid free in
solution (top) and sequestered (bottom).
[0038] FIG. 2A-C is a general diagram of an embodiment in which a
capture system, linked to the support via the interactor with the
label binding to the captured nucleic acid, is used.
[0039] FIG. 3A-C is a general diagram of an embodiment in which a
capture system, linked to the support via the label, is used.
[0040] FIG. 4A-B contain a graph illustrating the variation of
fluorescence signal intensity with DNA segment length (in base
pairs). Several sequences of variable lengths were tested, at the
same concentration (20 ng/ml), with either A) Picogreen or B) Popo3
as intercalating agent. Fluorescence using a single interactor to
bind the nucleic acid appears to be length dependent.
[0041] FIG. 5A-E are plots of fluorescence vs concentration of DNA
for DNA of different base pair lengths. Each sequence (X1 to X5;
A-E) is tested, with Picogreen, at several concentrations. In each
case, fluorescence is well correlated with concentration.
[0042] FIG. 6 is a plot of fluorescence vs. sequence identity.
Several different sequences of comparable lengths were tested, at
the same concentration (20 ng/ml), with a single intercalating
agent: either Picogreen .diamond-solid. or Popo3 .box-solid..
Fluorescence using a single interactor appears to be sequence
dependent.
[0043] FIG. 7 A-B are respectively a plot of fluorescence vs DNA
segment length (panel A) or sequence type (panel B) Several
sequences of A) variable lengths or B) variable sequence identity,
were tested, at the same concentration (20 ng/ml), with a mix of
four intercalating agents: picogreen, popo 3, yoyo 1, orange
acridin, at equimolar ratios. Fluorescence appears to be no longer
length- or sequence-dependent.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Definitions
[0044] A "support," for use with the present invention, is any
solid phase which can serve as a substrate for immobilizing the
first nucleic acid capture system. In some embodiments, the support
is the wall or a well, e.g., a well in a 96-well plate, or a wall
of a well fabricated into a glass or quartz slide. In certain
embodiments, the support is a tip. In other embodiments the support
is glass or quartz, e.g., a microscope slide, coverslip, or a
portion thereof. In some embodiments, the support is a particle,
bead or molecule that can be concentrated in solution (e.g.,
paramagnetic particles, streptavidin . . . ). In other embodiments,
the support is an inner wall of a reaction vessel. In particular
embodiments the support is paper, cellulose or nitrocellulose, or
any derivatives. In short any type of solid support can be
used.
[0045] A "vessel," for use with the present invention, can be any
tube (e.g., 0.2 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL in volume),
container, or well, which has an opening and is enclosed on all
other sides. Multiple wells can be joined together to form a plate,
such as a 96-well plate or any other plate adapted to be used in an
automated equipment. In terms of the present invention, a support
consisting of glass or quartz (as described above) is also
considered a vessel. In some embodiments the vessel is a tip.
[0046] A "linker," or "linking moiety," for use with the present
invention, is any molecule, set of molecules (e.g., binding
partners, such as biotin/streptavidin, antibody/antigen), or
chemical bond, which has served to connect either (1) the support
and the first capture system, (2) each of the first and second
capture system with the label (sandwich assay embodiments), (3) a
competing nucleic acid and a label (competitive method
embodiments), or (4) a label and a support (one labeled capture
system method). In some embodiments, the linker is a peptide, a
single stranded nucleic acid, a carbon chain, or simply a covalent
bond. In other embodiments, the linker is an antibody or nucleic
acid binding molecule. In some embodiments, the linker is composed
of a plurality of molecules.
[0047] A "linking entity", as used herein, is one of the units that
is attached to the linker. For example, a linker can have a support
and capture system as linking entities.
[0048] A "target" or "target nucleic acid", as used herein, is a
nucleic acid molecule to be isolated and quantified by the present
invention, and is present in a sample. The target can be either
single or double stranded nucleic acid. In some embodiments, the
target is cell-free nucleic acid such as DNA, and in some more
specific embodiments double-stranded cell-free DNA.
[0049] The term "nucleic acid", as used herein, encompasses both
single-stranded and double-stranded nucleic acid molecules. The
"nucleic acid" can be a DNA or RNA molecule, as well as a DNA/RNA
chimera, a nucleic acid molecule with unnatural bases and/or sugar
moieties, or a nucleic acid within another kind of molecule
chimera. The nucleic acid, when double stranded, has a minimum
length of 10 base pairs. In the case of single stranded molecules,
10 nucleotides is the minimum length.
[0050] "Cell free DNA", as used herein, is a DNA molecule
originally present within the nucleus of an intact cell, which is
free in an extracellular environment, after spontaneous or induced
cell lysis or disruption. In some embodiments, "cell free DNA" is
extracted, purified or amplified nucleic acid.
[0051] A "genome equivalent", as used herein, is the amount of DNA
originally present in one intact cell.
[0052] The "sample" used in the present invention can be any medium
which contains the target nucleic acid in a non-cellular (i.e., an
extracellular or other cell-free) environment. For example, blood,
plasma or serum or any other biological sample (e.g., CSF, urine)
can be used as samples for the present invention. In some
embodiments, the sample is a cell lysate. In some embodiments,
samples may be obtained from a purification, concentration,
amplification or dilution process. In some embodiments, samples may
be man-made, such as a media effluent from an industrial process.
In other embodiments, the sample is a mix of natural and man-made
fluids. In various embodiments, samples may be beverages, perfumes,
food, or any type of other fluid that could contain free nucleic
acids.
[0053] A "nucleic acid interactor" or "nucleic acid interacting
molecule", as used herein, refers to a molecule that binds to
nucleic acids with specificity to ensure subsequent steps of the
process. The interactor is therefore capable of capturing and/or
isolating (i.e., sequestering) a target nucleic acid from a sample.
Nucleic acid interactors include, without limitation, nucleic acid
intercalating agents (also referred to as intercalators), as well
as minor and major groove binders, small molecules which bind
nucleic acid in a non-sequence-specific manner,
non-sequence-specific nucleic acids, fusion molecules resulting
from a combination of several molecules or from a set of molecules
and single stranded nucleic acids (e.g., peptide-nucleic acid
chimera) and antibodies, or any type of other combination. These
molecules are used as the capture system to initially sequester the
target nucleic acid. In some embodiments of the invention, a
nucleic acid interactor can act as both an intercalating agent and
a minor or major groove binder.
[0054] In various embodiments, the "nucleic acid interactor"
results from a combination of nucleic acid interactors, to
compensate the sequence- or length-dependence of the interaction
between the specific interactor and the target nucleic acid. The
set of interactors ensures measurement of all nucleic acids
contained in the medium.
[0055] A "conjugate" or "conjugated nucleic acid", as used herein,
is a target nucleic acid bound to a nucleic acid interactor. In
various embodiments, a conjugated nucleic acid is a double stranded
nucleic acid or triple stranded nucleic acid bound to a nucleic
acid interactor. In embodiments where the target nucleic acid is
single stranded, a conjugate can refer to the double stranded
sequestered molecule, where one strand is the target molecule and
the other strand is part of the capture system or permits the
capture system to operate by forming a double stranded
conjugate.
[0056] A "sequestered conjugate", as used herein, refers to a
target nucleic acid bound to a nucleic acid interactor, wherein the
conjugate is also bound to a support.
[0057] A "capture system" or "capture molecule," as used herein, is
a compound or set of compounds used to bind and/or sequester the
target nucleic acid. The present invention makes use of at least a
first capture system, to sequester the target nucleic acid (herein,
"capture molecule 1," "capture system 1" or "first capture
system"). In the sandwich method, a second capture system is
employed (herein, "capture molecule 2", "capture system 2" or
"second capture system") to further bind the sequestered nucleic
acid. Capture system 2 is used for the sandwich assay embodiment
and is labeled with a readable molecule directly or through a
linker. In some embodiments, the second capture system is linked to
a molecule that may react with a binding partner linked to a label
directly or through a linker.
[0058] In some embodiments, a capture system (first or second
capture system) can include as a component a single stranded or a
set of single stranded non sequence-specific nucleic acid intended
to form a double stranded nucleic acid or triple stranded nucleic
acid with the target, a nucleic acid interactor, a nucleic acid
binding molecule such as a small molecule or a transcription
factor, or a chromosomal protein or a combination of them. In
certain embodiments, the capture system may be labeled, directly or
indirectly, or not labeled. In various embodiments, the capture
system may become readable once linked to the conjugate or the
sequestered conjugate. In certain embodiments, DNA or RNA or more
generally nucleic acid primers or nucleic acids having sequences
complementary to the target nucleic acid (such that the recognition
is sequence-specific) are excluded from the capture system.
[0059] In various embodiments, the capture system binds the target
nucleic acid by non-covalent interactions. Some non-covalent
interactions that can be employed are hydrogen bonding,
cation-.pi., .pi.-.pi. interactions, ionic pairing, hydrophobic
interaction, dipole-dipole, dipole-induced-dipole, charge-dipole
and van Der Waals interactions. In certain embodiments, the capture
system comprises antibodies which bind epitopes common to most or
all double stranded nucleic acids. For example, antibodies
generated in patients with various autoimmune diseases can be
harnessed. See Tzioufas A G, Manoussakis M N, Drosos A A et al.
"Enzyme immunoassays for the detection of IgG and IgM anti-dsDNA
antibodies: clinical significance and specificity." Clin Exp
Rheumatol. 1987; 5:247-253, incorporated by reference in its
entirety.
[0060] A "label", as used in the present invention, is any moiety
which can be used in the readout reaction and which binds the
target nucleic acid or competing nucleic acid, through a linker
(competitive assay) or through an additional capture system and
linker (sandwich assay). In certain embodiments the label may be
linked, directly or indirectly to the first capture system.
[0061] In certain embodiments, in which luminescence is used as the
readout method, the label will be the moiety from which light is
emitted. In some embodiments the label can be acridinium,
ruthenium, Europium, XL665 or dioxetane.
[0062] In various embodiments, the label is a particle detectable
in a magnetic field (e.g. magnetic, ferromagnetic paramagnetic or
superparamagnetic beads (Dynabeads)).
[0063] The signal emitted by the label can be optic (UV, Visible,
IR absorption), fluorescent, luminescent or magnetic, it may be
detected without limitations by NMR, circular dichroism, or Raman
spectroscopy.
[0064] In certain embodiments, the label is an enzyme that modifies
its substrate so as to be detected by any means cited supra,
including luminescence.
[0065] A "labeled conjugate", as used herein, is a conjugate,
sequestered or otherwise, further bound in some manner to a label.
A "sequestered labeled conjugate," refers to a labeled conjugate
further bound to a support.
[0066] "Essentially exclusively labeling a sequestered conjugate",
as used herein, means binding a label to a sequestered conjugate,
directly or indirectly. Non-specific binding of the label to
components other than sequestered conjugates may occur, but the
signal generated from these interactions can be subtracted from the
total signal and, therefore, false positive signals will not be
counted. The non-specific labeling can be quantified by a
calibration step before the method is carried out. In some
embodiments, the ratio of the label bound to a sequestered
conjugate as compared to other components in the reaction is
1000:1, 500:1, 100:1 or 10:1. In some embodiments, the ratio is 5:1
or 2:1.
[0067] In some embodiments, when a conjugate is "essentially
exclusively labeled," the labeling starts in solution, i.e., the
support is not labeled with the readable molecule prior to the
addition of target nucleic acid (the support is not "prelabeled").
The earliest the label can associate (i.e., indirectly bind, see
FIG. 1A) with the support is at the time the target nucleic acid
binds the first capture system. The first capture system is the
reaction component that will either be bound to the support
originally (see FIG. 1A), or will bind the support after
introduction into the reaction. The label cannot indirectly bind
the support without the first capture system and the nucleic acid
molecule (see FIG. 1A).
[0068] In some embodiments, the label may be linked, directly or
indirectly, to the first capture system and link itself to the
support, directly or indirectly. In this case, the capture system
binds the nucleic acid, in a non-sequence-specific manner, and
there is no need for a second step to bind the label, already
linked, via the capture system, to the conjugate. In certain
embodiments, the entity formed by the capture system and the label
may become readable once the capture system binds to the target, or
once the label links itself to the conjugate (see FIG. 1C)
[0069] In some embodiments, the label may be linked, directly or
indirectly, to the support. It may be linked, directly or
indirectly, to the capture system. In this case the capture system
binds the nucleic acid, in a non-sequence-specific manner, and
there is no need for a second step to bind the label, already
linked, via the capture system, to the conjugate. In certain
embodiments the entity formed by the capture system and the label
may become readable once capture system binds to the target, or
once the label links itself to the conjugate (see FIG. 1D)
[0070] The term "particles detectable in a magnetic field" or
"magnetic particles", as used herein, is intended to encompass all
particles (e.g., beads or irregular shape particles) that can
capture (i.e., interact with, or associate with) cells or nucleic
acids or both (under different buffer conditions). Included are
ferromagnetic, paramagnetic, and superparamagnetic particles, which
are commercially available through companies such as Promega and
Invitrogen.
[0071] A "competitive assay" or "competitive method", as used
herein, is an assay in which a known concentration of labeled
nucleic acid molecules competes with the target nucleic acid
(present in the sample) for binding to the capture system. The
amount of label detected is inversely proportional to the amount of
target nucleic acid originally present in the sample. See U.S. Pat.
No. 5,198,368 incorporated herein by reference, for a detailed
description of competitive fluorescence/luminescence assays.
[0072] A "sandwich assay" or "sandwich method", as used herein, is
an assay in which target nucleic acid binds to the capture system
1, therefore sequestering the nucleic acid to the support. The
target nucleic acid is quantified by binding a second capture
system to the sequestered nucleic acid, which is labeled, and by
detecting the quantity of label.
[0073] A "one labeled capture system method", as used herein, is an
assay in which the first capture system contains a label. Once the
target nucleic acid is bound the sequestered capture system, the
label becomes active. The amount of the label detected is
proportional to the amount of target nucleic acid present in the
sample.
[0074] A "luminescent reaction", as used herein, is any reaction in
which light is produced. There may or may not be an electron
excitation step, by light directly or by fluorescence resonance
energy transfer (FRET, see e.g., Patent No WO 2010/129787), to
generate the light (e.g., a fluorescence based assay). When no
electron excitation takes place, light is generated by either a
chemiluminescent or a bioluminescent reaction (e.g.,
luciferase-mediated oxidation, acridium ester with hydrogen
peroxide, alkaline phosphatase with dioxetane, dioxetane with
.beta.-galactosidase, horseradish peroxidase with luminol and gold
mixed with luminol, phosphatase and ombelliferone, electrochemical
excitation of ruthenium and tripropylamine, or UV excitation of
Europium).
Specific Embodiments
[0075] By way of overview and introduction, FIGS. 1A and 1B provide
schematics of two classes of embodiments of the invention. FIG. 1A
represents the sandwich assay embodiment. A reaction vessel 100
contains a support 10, linked to a first capture system e.g., (a
first intercalator or other nucleic acid interactor 30 through a
linker 20). The support can be, for example, the inner vessel wall
or a magnetic particle. The first capture system 30 discriminately
sequesters the target nucleic acid 40 from a crude sample 60,
containing impurities. In some embodiments, a second capture system
30' (i.e., a second nucleic acid interactor) is added to the
reaction and binds the sequestered target nucleic acid. The second
capture system 30' is linked to a readable label 50 through a
linker 20'.
[0076] FIG. 1B shows a schematic of a general competitive assay
embodiment of the invention. A reaction vessel 100 contains a
support 10, linked to a first capture system 30 through a linker
20. The support can be, for example, the inner vessel wall or a
magnetic particle. The capture system 30 discriminately sequesters
the target nucleic acid 40 from a crude sample 60, containing
impurities. The target nucleic acid 40 competes with a known
concentration of labeled nucleic acid 40' for binding to the
capture system. The known quantity of labeled nucleic acid 40' is
linked to a readable label 50'' through a linker 20''.
[0077] FIG. 2 panel A shows a schematic of a general simplified
method using a labeled capture system embodiment of the invention.
A reaction vessel 100 contains a support 10, linked to a first
capture system 30 through a linker 20. The support can be, for
example, the inner vessel wall or a magnetic particle. In panel B,
the capture system discriminately sequesters the target nucleic
acid 40 from a crude sample 60, containing impurities. In Panel C,
the label 50'' linked to the capture system through a linker 20'''
interacts with the conjugate formed by capture system 30 and the
target nucleic acid 40.
[0078] FIG. 3 shows a schematic of a general simplified method
using a labeled capture system embodiment of the invention. In
Panel A, a reaction vessel 100, contains a support 10, linked to a
label 50'' through a linker 20. In Panel B, the capture system 30,
linked to the label 50' through a linker 20''', discriminately
sequesters the target nucleic acid 40 from a crude sample 60,
containing impurities. The support can be, for example, the inner
vessel wall or a magnetic particle. In Panel C, the label 50''
interacts with the conjugate formed by the capture system 30 and
the target nucleic acid 40. The components of the three assays are
discussed below.
[0079] Target Nucleic Acid
[0080] The process of the invention begins with providing a sample
60 containing the target nucleic acid 40, which is to be isolated
and quantified. In some embodiments, the sample 60 is crude blood,
plasma, serum, urine or cerebrospinal fluid. In certain
embodiments, the sample is a cell lysate.
[0081] In some embodiments, samples may be obtained from biological
samples such as blood or other bodily fluids, pursuant to a
purification, concentration, amplification or dilution process. In
some embodiments, samples may be man-made, such as a media effluent
from an industrial process. In other embodiments, the sample is a
mix of natural and man-made fluids. In various embodiments, samples
may be beverages, perfumes, food, or any type of other fluid that
could contain free nucleic acids.
[0082] In some embodiments, the sample 60 contains intact cells, as
well as extracellular nucleic acids. The present invention serves
to quantify the extracellular nucleic acids present in a sample and
does not quantify the nucleic acids present inside intact cells,
should cells also be present within a sample. When the sample is a
cell lysate, the invention can quantify double stranded nucleic
acids, single stranded nucleic acids, or both types of nucleic
acids. In samples where specific kinds of nucleic acids are
isolated from a pool of nucleic acids (e.g., mRNA molecules with
DNA and ribosomal RNA background), capture systems (discussed
infra) are designed to sequester the appropriate type of nucleic
acid.
[0083] In some embodiments, the target nucleic acid 40 is cell free
DNA and is present in a blood, plasma, serum or other biological
fluid sample. Alternatively, the target nucleic acid 40 can be
single stranded nucleic acid, such as mRNA. In certain embodiments,
the target is nuclear DNA present in a cell lysate.
[0084] In some embodiments, the target nucleic acid 40 is extracted
and/or purified, and/or amplified nucleic acid. In embodiments, the
nucleic acids are not labeled with a readable label.
[0085] Capture System
[0086] In some embodiments, a method provides contacting the target
nucleic acid in the sample with at least one nucleic acid
interactor to form a conjugate; and sequestering the conjugate by
reacting the conjugate with a capture molecule bound to a support.
A "capture system" or "capture molecule," as used herein, is a
compound or set of compounds used to bind and/or sequester the
target nucleic acid. The present invention makes use of at least a
first capture system, to sequester the target nucleic acid (herein,
"capture molecule 1," "capture system 1" or "first capture
system"). In the sandwich method, a second capture system is
employed (herein, "capture molecule 2", "capture system 2" or
"second capture system") to further bind the sequestered nucleic
acid. Capture system 2 is used for the sandwich assay embodiment
and is labeled with a readable molecule directly or through a
linker. In some embodiments, the second capture system is linked to
a molecule that may react with a binding partner linked to a label
directly or through a linker.
[0087] In embodiments, a first capture system for a target nucleic
acid comprises a support, a linker, and a capture molecule that
binds to nucleic acids. In embodiments, a first capture system
further comprises a readable label or signal generating element. In
embodiments, the readable label or signal generating element is
bound to the capture molecule and/or the linker.
[0088] In other embodiments, a second capture system comprises a
capture molecule that binds to nucleic acids, a linker, and a
signal generating element.
[0089] In some embodiments, a linker is selected that minimizes
steric hindrance and chemical interaction with the interactors.
[0090] In embodiments, the sample 60 is added to a reaction vessel
100 where the isolation and quantification reactions are performed.
In embodiments, the inner walls of the vessel, or a portion
thereof, can act as a support 10 for linking a first nucleic acid
capture system. In some embodiments, the support 10 is a portion of
a glass or quartz microscope slide, and can be a well fabricated in
the slide or a well in a plate. In various embodiments, the support
10 is a concentratable particle, which can include a bead/particle,
for example, a magnetic particle. In various particle embodiments,
the particle is covalently attached to the first capture system. In
some embodiments, the particle (immobilized or support) is
pre-derivatized with the first capture system 30.
[0091] The derivatization can occur through a linker 20, which can
be a direct covalent bond between the capture system 30 and the
support 10. The linker can also be a single type of molecule or a
plurality of different molecules. The linker may contain a specific
binding pair (e.g., avidin-biotin, antibody-antigen,
enzyme-substrate). In a specific binding pair embodiment, one of
the specific pair members is directly bound to the particle and
will bind the capture system, by binding or interacting with the
partner molecule, which is in turn bound to a nucleic acid
interactor (i.e., capture system 30).
[0092] Depending on what molecule for the linker 20 is used, it
will be apparent to one of ordinary skill in the art as to how to
attach the linker to the substrate. For example, a detailed
discussion on attaching biotin to glass substrates is provided in
U.S. Pat. No. 5,919,523, incorporated by reference in its
entirety.
[0093] The target nucleic acid is isolated from the sample with the
use of a first capture system. The first capture system 30 is
attached to the support via a linker. The first capture system, in
certain embodiments, discriminately isolates double stranded
nucleic acids from a sample that contains other impurities and/or
single stranded nucleic acids.
[0094] In some embodiments, a capture system (first or second
capture system) can include as a capture molecule a single stranded
or a set of single stranded non sequence-specific nucleic acid
(such as for example poly T to bind up poly A of mRNA or poly GC,
if there is repetitive DNA--such as CpG islands) intended to form a
double stranded nucleic acid or triple stranded nucleic acid with
the target, a nucleic acid interactor, and a nucleic acid binding
molecule such as a small molecule or a transcription factor, or a
chromosomal protein or a combination of them. In certain
embodiments, the capture system may be labeled, directly or
indirectly, or not labeled. In various embodiments, the capture
system may become readable once linked to the conjugate or the
sequestered conjugate. In certain embodiments, nucleic acid primers
or nucleic acids having sequences complementary to the target
nucleic acid (such that the recognition is sequence-specific) are
excluded from the capture system.
[0095] In various embodiments, the capture system binds the target
nucleic acid by non-covalent interactions. Some non-covalent
interactions that can be employed are hydrogen bonding,
cation-.pi., .pi.-.pi. interactions, ionic pairing, hydrophobic
interaction, dipole-dipole, dipole-induced-dipole, charge-dipole
and van Der Waals interactions. In certain embodiments, the capture
system comprises antibodies which bind epitopes common to most or
all double stranded nucleic acids. For example, antibodies
generated in patients with various autoimmune diseases can be
harnessed. See Tzioufas A G, Manoussakis M N, Drosos A A et al.
"Enzyme immunoassays for the detection of IgG and IgM anti-dsDNA
antibodies: clinical significance and specificity." Clin Exp
Rheumatol. 1987; 5:247-253, incorporated by reference in its
entirety.
[0096] In the present invention, a nucleic acid interactor or in a
preferred embodiment, an intercalator is used as a capture molecule
30. The interactor can be bound to a specific binding pair member
wherein the other member is covalently attached to the support 10,
for example, a paramagnetic particle. The sample 60 is added to the
reaction mixture and mixed with the interactor-derivatized
particle. The particle can then be sequestered from the sample by
applying a magnetic field to the vessel and decanting the liquid
phase.
[0097] A "nucleic acid interactor" or "nucleic acid interacting
molecule", as used herein, refers to a molecule that binds to
nucleic acids with specificity to ensure subsequent steps of the
process. The interactor is therefore capable of capturing and/or
isolating (i.e., sequestering) a target nucleic acid from a sample.
Nucleic acid interactors include, without limitation, nucleic acid
intercalating agents (also referred to as intercalators), as well
as minor and major groove binders, small molecules which bind
nucleic acid in a non-sequence-specific manner,
non-sequence-specific nucleic acids, fusion molecules resulting
from a combination of several molecules or from a set of molecules
and single stranded nucleic acids (e.g., peptide-nucleic acid
chimera) and antibodies, or any type of other combination. These
molecules are used as the capture system to initially sequester the
target nucleic acid. In some embodiments of the invention, a
nucleic acid interactor can act as both an intercalating agent and
a minor or major groove binder. In embodiment, the nucleic acid
interactor does not permeate a cell.
[0098] In certain embodiments, the support 10 is covalently bound
to the first capture system 30. In these embodiments, the covalent
bond between the support and capture system is the linker 20. In
some embodiments, the support is covalently bound to a linker
molecule which binds the capture system either covalently or
non-covalently.
[0099] In embodiments, nucleic acid interactors are harnessed as
the main component of the first capture system 30 and/or second
capture system 30'. In some embodiments, the capture molecule in
the first capture system and the second capture system are
different from one another. In other embodiments, the capture
molecule in the first capture system and the second capture system
are the same. In some cases, when two or more different nucleic
acid interactors are used only one of the nucleic acid interactors
is bound to a support. In yet other cases, when two or more nucleic
acid interactors are used one of the nucleic acid interactors is
bound to a support, a different nucleic acid interactor is bound to
a label. In some embodiments, two or more nucleic acid interactors
are selected to have similar spectral properties. In some other
embodiments, two or more nucleic acid interactors are selected to
bind to different types of nucleic acids or bind at a different
structural feature of a nucleic acid.
[0100] The interactors, such as DNA intercalators, can be bound to
a substrate (e.g., beads) through a linker 20 or 20', which can be
a linking molecule or simply a covalent bond. Suitable linkers for
intercalator embodiments are known; some are, for example,
described in U.S. Pat. No. 4,921,805, incorporated herein by
reference.
[0101] A "linker," or "linking moiety," for use with the present
invention, is any molecule, set of molecules (e.g., binding
partners, such as biotin/streptavidin, antibody/antigen), or
chemical bond, which has served to connect either (1) the support
and the first capture molecule, (2) each of the first and second
capture molecules with the label (sandwich assay embodiments), (3)
a competing nucleic acid and a label (competitive method
embodiments), or (4) a label and a support (one labeled capture
system method). In some embodiments, the linker is a peptide, a
single stranded nucleic acid, a carbon chain, or simply a covalent
bond. In other embodiments, the linker is an antibody or nucleic
acid binding molecule. In some embodiments, the linker is composed
of a plurality of molecules.
[0102] In some embodiments, the capture molecule and the solid
support 10 are first modified in such a way that they can be joined
together through an amide, carbamate, urea, ether, thioether, amine
or other linkage commonly used for immobilization (Affinity
Chromatography. Hoffman-Osterhof, ed.; Pergamon Press, 1978;
Affinity Chromatography, publication of Pharmacia Fine Chemicals).
For example, if the solid support 10 is modified to contain a
carboxy group, then the intercalator or interactor may be modified
to contain a reactive amine through addition of a spermine or
diaminoalkane molecule. Using coupling agents such as
dicyclohexylcarbodiimide, the support and the intercalator can be
joined together through an amide bond. Alternatively, the carboxyl
group can be converted to a reactive ester (such as
N-hydroxysuccinimide ester) which will then react with the amine.
Conversely, the support can be modified with an amine and the
nucleic acid interactor can be modified to contain a carboxyl group
and the two moieties coupled together as described above.
[0103] The linkers 20, 20' and 20'', can be covalent bonds, as
described above, or can be a molecule or plurality of molecules,
such as specific binding pairs. The linker molecule may be charged
or uncharged, and in certain embodiments, comprises methylene
groups joined together with amide bonds (see, e.g., U.S. Pat. No.
4,921,805).
[0104] The linkers 20, 20', 20'' and 20''' of the present
invention, must be bifunctional in the sense that it can bind to
both the support and first capture system (20) or to both the
second capture system and the label (20') or to both the competing
nucleic acid and the label (20'') or to both the first capture
system and the label (20''').
[0105] In certain embodiments, the linker is the shortest path
connecting the two linkable entities (i.e., capture system 1 and
support (20), capture system 2 and label (20'), competing nucleic
acid and label (20''), capture system 1 and label (20''')). A
linker, in some embodiments, is a chain of atoms or a branched
chain of atoms. Chains can be saturated as well as unsaturated. The
linker may also be a ring structure with or without conjugated
bonds. It can be a molecule that consists of alkyl, alkenyl,
alkynyl or aryl groups, as well as any combination of these groups.
The linker can also comprise a fluorescent substrate, for use in
FRET assays.
[0106] In some embodiments, the linker 20 is substituted by any
chemical group with one or more atoms selected from the group
consisting of C, H, O, S, N, P, Se, Ge, Sn and Pb. For example, the
linker 20 may comprise a chain of "m" atoms selected from the group
consisting of C, O, S, N, P, Se, Ge, Sn, Pb, wherein one end of the
chain is connected to the first entity (i.e., support, capture
system 2, or competing nucleic acid) and the other end of the chain
is connected to the second entity (i.e., capture system 1 or
label). In various illustrative embodiments, the linker 20 can be a
bond, a specific binding pair, a peptide, a pseudopeptide, a
nucleotide or a pseudonucleotide, a polysaccharide, and can either
be branched or linear. The linker molecule may or may not be
substituted.
[0107] The total length of the linker 20 and its chemical structure
are adjusted depending on the size and the chemical structures of
the nucleic acid interacting molecule (e.g., intercalating agent)
and linking entities. The optimum adjustment of these parameters
can be achieved (based in part on the type of linker) by molecular
modelling by using an empirical force field like CHARMM (Brooks, B.
R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan,
S., and Karplus, M. (1983) J. Comput. Chem. 4, 187-212) defining
solvent mimicking the in vitro environment, taking into account
concentration of salt and buffer, and the pH of the solution.
Empirical testing can also be employed to determine optimal linker
properties.
[0108] A preferred linker for a specific couple of linking entities
can be designed to: (1) be energetically stable in a range of pH
and temperature values compatible with the reaction performed for
DNA quantification (e.g., the luminescent reaction or magnetic
detection) and avoid degradation in a range of pH and temperature
values compatible with the biochemical reaction performed for DNA
quantification.
[0109] In preferred embodiments, the linker 20 is energetically
stable and non-degradable for at least a period of time from the
beginning of the assay (capture of target) through completion of
the time needed until performing the readout reaction, as well as
when subjected to the physical conditions and environment (e.g.,
pressure, hygrometry, vibrations, pollution, temperature, noise,
light, agitation, etc.) needed to carry out the reaction. Linker
stability should also not be affected by dilution.
[0110] The linker 20 can be inert with the rest of the constituents
of the reagents' medium and should be soluble in the medium where
the assay of the present invention is conducted.
[0111] In addition, the linker 20, in preferred embodiments, does
not adopt a conformation that sterically interferes with the
readout reaction or with the nucleic acid capture. The linker 20
also avoids the alteration of the folding and/or the chemical
structure of the support 10 and of the nucleic acid interactor
(e.g., intercalating agent) by, for example, electron
delocalization, radical generation, electronic modifications
leading to a loss of intercalating, interacting, magnetic or optic
or luminescent properties, or a loss of integrity of the support
leading to a release of the linker/intercalating agent couple from
the surface of this sited support.
[0112] In some embodiments, the linker 20 is compatible for
additional conjugated chemical reactions in the medium (e.g.,
binding an antibody specific for one member of the
support/linker/intercalating agent/target molecule complex, without
altering the structure and stability of the complex, except if this
effect is needed for the correct achievement of the reaction).
[0113] Importantly, the linker 20 should have a chemical structure
compatible with the attachment with one or more nucleic acid
interacting molecules and support.
[0114] In some embodiments, a first and/or second capture system
comprises a readable label. In some embodiments, the readable label
is bound to the linker and in others, to the capture molecule. A
"label", as used in the present invention, is any moiety which can
be used in the readout reaction and which binds the target nucleic
acid or competing nucleic acid, through a linker (competitive
assay) or through an additional capture system and linker (sandwich
assay). Multiple labels may be employed in order to identify
different types of nucleic acids, e.g. double stranded DNA, mRNA,
and the like.
[0115] In some embodiments, the label may be linked, directly or
indirectly, to the first capture system and link itself to the
support, directly or indirectly. In this case, the capture system
binds the nucleic acid, in a non-sequence-specific manner, and
there is no need for a second step to bind the label, already
linked, via the capture system, to the conjugate. In certain
embodiments, the entity formed by the capture system and the label
may become readable once the capture system binds to the target, or
once the label links itself to the conjugate (see FIG. 1C).
[0116] In some embodiments, the label may be linked, directly or
indirectly, to the support. It may be linked, directly or
indirectly, to the capture system. In this case the capture system
binds the nucleic acid, in a non-sequence-specific manner, and
there is no need for a second step to bind the label, already
linked, via the capture system, to the conjugate. In certain
embodiments the entity formed by the capture system and the label
may become readable once capture system binds to the target, or
once the label links itself to the conjugate (see FIG. 1D).
[0117] In certain embodiments, in which luminescence is used as the
readout method, the label will be the moiety from which light is
emitted. In some embodiments the label can be acridinium,
ruthenium, Europium, XL665 or dioxetane.
[0118] In various embodiments, the label is a particle detectable
in a magnetic field (e.g. magnetic, ferromagnetic paramagnetic or
superparamagnetic beads (Dynabeads)).
[0119] The signal emitted by the label can be optic (UV, Visible,
IR absorption), fluorescent, luminescent or magnetic, it may be
detected without limitations by NMR, circular dichroism or Raman
spectroscopy.
[0120] In certain embodiments, the label is an enzyme that modifies
its substrate so as to be detected by any means cited supra,
including luminescence.
[0121] A "labeled conjugate", as used herein, is a conjugate,
sequestered or otherwise, further bound in some manner to a label.
A "sequestered labeled conjugate," refers to a labeled conjugate
further bound to a support.
[0122] "Essentially exclusively labeling a sequestered conjugate",
as used herein, means binding a label to a sequestered conjugate,
directly or indirectly. Non-specific binding of the label to
components other than sequestered conjugates may occur, but the
signal generated from these interactions can be subtracted from the
total signal and, therefore, false positive signals will not be
counted. The non-specific labeling can be quantified by a
calibration step before the method is carried out. In some
embodiments, the ratio of the label bound to a sequestered
conjugate as compared to other components in the reaction is
1000:1, 500:1, 100:1 or 10:1. In some embodiments, the ratio is 5:1
or 2:1.
[0123] In the present invention, when a conjugate is "essentially
exclusively labeled," the labeling starts in solution, i.e., the
support is not labeled with the readable molecule prior to the
addition of target nucleic acid (the support is not "prelabeled").
The earliest the label can associate (i.e., indirectly bind, see
FIG. 1A) with the support is at the time the target nucleic acid
binds the first capture system. The first capture system is the
reaction component that will either be bound to the support
originally (see FIG. 1A), or will bind the support after
introduction into the reaction. The label cannot indirectly bind
the support without the first capture system and the nucleic acid
molecule (see FIG. 1A).
[0124] The first capture system 30 can be any molecule or
molecules, attached to the support 10 via a linker 20, and serves
to isolate the desired nucleic acid from a crude sample (i.e., a
sample that contains undesirable nucleic acids and/or non-nucleic
acid components). In a preferred embodiment, the first capture
system comprises a nucleic acid intercalating agent.
[0125] Based on the capture system 30 chosen, it will be apparent
to one of ordinary skill in the art, how to immobilize the capture
system or in any event attach it to the support.
[0126] In some embodiments, the support 10 will be prederivatized
with the capture system 30. In other embodiments, the support 10
will sequester or bind the capture system 30 when the user combines
the two in a reaction, e.g., by use of specific binding pairs.
[0127] In various embodiments, the target nucleic acid 40 to be
detected is a single stranded molecule. When this is the case, the
first capture system 30 can be comprised of a molecule that binds,
in a non-sequence-specific manner, the target based on secondary
structure. In other embodiments small molecules which bind
universal stretches of nucleic acids can be used to sequester
target nucleic acids from the sample. In the small molecule
embodiments, multiple small molecules can be harnessed, in order to
bind all possible nucleotide stretches. This can be accomplished by
derivatizing the support with a library of small molecules.
Although single stranded nucleic acids can be isolated and
quantified from a crude sample by the methods presented herein, a
preferred target is a double stranded nucleic acid. When the target
is double stranded, a preferred capture system 30 is a set of
nucleic acid intercalators. The present inventors have shown that
the two intercalators help eliminate or significantly reduce
measurement dependency on target size and/or target sequence.
[0128] In various embodiments, one or a set of single stranded non
sequence-specific nucleic acids, can be used as a portion of the
first capture system 30 and can sequester, for example, target
nucleic acid 40 from the sample 60 to form a double helix, or
target double stranded nucleic acid 40 to form a triple helix. In
these embodiments, a nucleic acid intercalator is not necessary for
use in the first capture system 30, although a combination of
capture molecules (e.g., single stranded nucleic acid &
intercalating agent), or fusion molecules, can be used in
combination as first capture systems, in the same reaction.
[0129] In certain embodiments, wherein the target nucleic acid 40
is single stranded, double stranded molecules are made by adding
single stranded nucleic acids to the reaction before sequestration
of the single stranded target nucleic acid 40 by the first capture
system 30. In these embodiments, careful consideration is paid to
the sequences of the additional single stranded nucleic acids, as
the formation of non-specific double helices will lead to false
positives in the quantification step. In some embodiments, the
single stranded nucleic acid molecules added to the sample will be
part of a fusion molecule, resulting from a combination of several
molecules or molecules and single stranded nucleic acid (e.g.,
peptide-nucleic acid chimera). In these embodiments, the fusion
molecule can be considered a portion of the first capture system
30. In other embodiments, a nucleic acid intercalator is used as
the capture system 30 to sequester the newly formed double stranded
molecules from solution.
[0130] In other embodiments, the single stranded, or a set of
single stranded non sequence-specific nucleic acid complements, can
be used as a capture system 30.
[0131] In some embodiments, the first capture molecule 30 can be an
antibody, a portion thereof, or a protein other than an antibody,
which will recognize the peptide portion of the fusion molecule
(described supra). Alternatively, a capture system 30 can be
comprised of a protein that can recognize stretches of DNA, e.g., a
transcription factor. In some embodiments, the first capture system
30 (and/or second capture system 30', depending on whether a
sandwich assay is employed) is a nucleic acid binding molecule,
such as a transcription factor or histone protein, which will
specifically bind a target nucleic acid 40. Small (natural or
synthetic) molecules can also be used as a capture system 30. For
example, molecules disclosed in DNA and RNA Binders, From Small
Molecules to Drugs, Martine Demeunynck, Wiley-VCH (2003) and Nelson
et al. Mutat Res., V. 623, pp. 24-40 (2007), can be used as capture
molecules of either the first 30 or second 30' capture system.
[0132] In certain embodiments, the first (30) or second (30')
capture system is an antibody or a library of antibodies specific
for epitopes present on the target nucleic acid 40. The second
capture system 30' can be an antibody, a plurality of antibodies
(e.g., library of antibodies specific for epitopes present on the
target nucleic acid), or epitopes spanning the first capture system
30 and the target nucleic acid 40.
[0133] In other embodiments, the capture system 30 comprises one or
a plurality of polymers which can bind either nucleic acids or
intercalating agents, or both, such as the ones disclosed in U.S.
Published Application No. 2004/0157217, incorporated herein by
reference in its entirety.
[0134] The first 30 and second 30' capture system can make use of
non-covalent interactions to bind the free target nucleic acid or
sequestered nucleic acid. Some non-covalent interactions that can
be employed are hydrogen bonding, cation-.pi., .pi.-.pi.
interactions, ionic pairing, hydrophobic interaction,
dipole-dipole, dipole-induced-dipole, charge-dipole and van Der
Waals interactions.
[0135] The capture system 30 or 30' may comprise a molecule or
several molecules. It reacts with nucleic acid by establishing
covalent or non-covalent interactions with the target nucleic acid
40, or in the case of the second capture system 30', interactions
can be between the capture system and epitopes spanning the first
capture system 30 and target nucleic acid 40. Specific capture
systems that may be employed include, but are not limited to
intercalating agents, cyanine dimers, cell impermeant cyanine
monomers, minor or major groove linkers, orange acridin, 7-AAD, LDS
751, and hydroxystilbamidin. Such agents are described in Chapter 8
of The Handbook--A Guide to Fluorescent Probes and Labeling
Technologies, 11.sup.th Edition, published by Molecular Probes) and
incorporated by reference in its entirety.
[0136] In various embodiments, an intercalating agent for use as a
capture system 30, or a component of a capture system, can be,
without limitation, for example, echinomycin, ethidium, ethidium
bromide, propidium bromide, methidium, acridine, aminoacridine,
quinacrin, acridine orange and derivatives thereof, psoralen,
proflavin, ellipticine, actinomycin D, daunomycin, malachite green,
phenyl neutral red, mitomycin C, Hoechst33342, Hoechst33258,
aclarubicin, DAPI, SYBR, Picogreen, Adriamycin, pirarubicin,
actinomycin, tris(phenanthroline) zinc salt, tris(phenanthroline)
ruthenium salt, tris(phenantroline) cobalt salt, di(phenanthroline)
zinc salt, di(phenanthroline) ruthenium salt, di(phenanthroline)
cobalt salt, bipyridine platinum salt, terpyridine platinum salt,
phenanthroline platinum salt tris(bipyridyl) zinc salt,
tris(bipyridyl) ruthenium salt, tris(bipyridyl) cobalt salt
di(bipyridyl) zinc salt, di(bipyridyl) ruthenium salt,
di(bipyridyl) cobalt salt, luzopeptin, triostin A, oxazole yellow,
thiazole orange TOTO, eth D., TOTA B, YOYO and derivatives and
dimers of the foregoing.
[0137] In some embodiments, a minor groove binder is used as a
nucleic acid interactor (and as a capture system 30). The minor
groove binder can be, without limitation, for example, Hoechst
33258, netropsin, distamycin, plicamycin, CDPI.sub.1-3,
lexitropsin, mithramycin, chromomycin A.sub.3, olivomycin,
anthramycin, sibriromycin, pentamidine, stilbamidine, berenil, or
any other minor groove binder.
[0138] Still, in other embodiments, a capture system 30 can employ
an interactor that acts as both a nucleic acid intercalator and a
(minor or major) groove binder. For the purposes of the assay, it
is irrelevant as to how the interactor sequesters the target
nucleic acid 40 from the remaining sample components, so long as
the target nucleic acid 40 is sequestered. Heterogeneous capture
systems that use both an intercalator and groove binder can also be
used as capture system 1 or 2 (30 or 30' respectively).
[0139] In other embodiments, the first capture system 30
selectively isolates target single stranded nucleic acid 40 from a
sample 60 that may have double stranded nucleic acid.
[0140] In some embodiments, reacting material may be transferred,
at any step, from one vessel to another, by automated process.
[0141] Sandwich Assays
[0142] The sandwich assay embodiment makes use of a second capture
system 30'. Once target nucleic acids 40 are sequestered from the
crude sample 60, the second part of the reaction may occur in the
same vessel or after automatic transfer by the machine, in another
one. This can be done immediately or after a wash or buffer
exchange step, to remove sample impurities. Wash steps can be
automated. Then, the second capture system 30' is added to the
reaction. When using particles as the support 10 for the first
capture system, the sample 60 (containing the target nucleic acid
40) is added to the reaction and mixed with the particles. The
particles (now bound to the target molecules) are sequestered by
applying a magnetic field. At this point, a wash/supernatant
removal step can be employed. The magnetic field is then
discontinued and a second capture system 30', labeled with a
readable molecule 50, is added to the reaction, optionally after a
wash step. After incubation, particles are sequestered. After the
wash step, the reading method is activated in the same vessel or
after automatic transfer by the machine, in another one.
[0143] Alternatively, the two capture systems (30 and 30') may be
added to the reaction simultaneously. After incubation with both
capture systems, excess capture systems are removed from the
reaction with a wash step. After the wash step, the reading method
is activated in the same vessel or after automatic transfer by the
machine, in another one.
[0144] If a capture system is bound to the wall of a reaction well
or vessel, automated or manual wash steps can be employed after
nucleic acid sequestration. These steps serve to remove impurities
from the original sample.
[0145] Once the target nucleic acid 40 is isolated/sequestered from
the sample 60, the user can proceed with detection (and
quantification) of the nucleic acid. In the case of the sandwich
assay, a second capture system 30' (which is labeled) is added to
the reaction. The second capture system 30' comprises a nucleic
acid interactor that will bind to the double, the triple stranded,
or to a free part of the single stranded sequestered nucleic acid,
or with the structure resulting from the interaction between
capture system 1 and nucleic acid. The two capture systems may be
added simultaneously or in a serial manner.
[0146] The sequestered nucleic acid is essentially exclusively
labeled with the readable molecule, and nonspecific labeling of
other reaction components is factored into the end signal. This is
accomplished through an initial calibration step.
[0147] The calibration step is carried out by measuring known
amounts of nucleic acids, subjected to the process of the present
invention. Increasing known amounts of nucleic acids are measured,
in order to generate a linear relationship between the detected
signal (e.g., light emitted) and nucleic acid molecules initially
present (also known as a standard curve). The linear relationship
has both an upper and lower limit, which establish the dynamic
range of the assay. The measured signal is proportional to the
amount of nucleic acid molecules initially present. Therefore, when
an unknown target nucleic acid 40 is subjected to the process
disclosed herein, the number of target nucleic acid molecules
initially present can be extrapolated from the standard curve, by
determining where along the curve the generated signal falls.
[0148] The amount of target nucleic acid molecules present in the
sample is then compared to a value that corresponds to the amount
of nucleic acids in a sample from a subject that does not have a
particular disease or disorder. For example, if a sample analyzed
from a subject suspected of having cancer has an amount of target
nucleic acid that is at least two fold greater, at least 4 fold
greater, at least 5 fold greater, or at least 10 to 100 fold
greater than that from a sample from a subject without cancer is
indicative of a pathological condition. In some embodiments, an
amount of cell free nucleic acid in the sample of at least about 5
ng/ml or at least 10 ng/ml is indicative of a pathological
state.
[0149] In embodiments, a result indicative of a pathological state
is then communicated to a health care provider. The health care
provider will order or administer one or more additional diagnostic
tests to detect the pathology. Such tests can include, tests for
one or more cancers including biomarker tests and biopsies, tests
for cardiac injury and/or liver injury by detecting elevated
cardiac or liver enzymes, and the like.
[0150] The second capture system 30' (e.g., a DNA binding protein,
interactor, antibody) is covalently or non-covalently bound to the
label 50 via a linker 20'. In various embodiments, the second
capture system 30' may become activated and then readable after
interaction with the conjugate.
[0151] Nucleic acid can be detected by methods known to those of
ordinary skill in the art. Luminescent labels can be read off by
harnessing fluorescence resonance energy transfer (FRET),
electronic or electrochemical excitation (to excite a fluorescent
label), or by chemiluminescence or bioluminescence reactions. In
some embodiments, the labeled capture system will bind
subpopulations of target nucleic acid 40 and are linked to distinct
labels to allow for multiplex detection.
[0152] Examples of luminescent reactions amenable for use with the
present invention are, without limitation, luciferase-mediated
oxidation, acridium ester with hydrogen peroxide, alkaline
phosphatase with dioxetane, dioxetane with .beta.-galactosidase,
horseradish peroxidase with luminol and gold mixed with luminol, or
electrochemical excitation of ruthenium and tripropylamine.
[0153] In some embodiments, luminescent detection is the detection
of a fluorescent substrate. The second capture system 30' is bound
via a linker to a fluorescent dye or molecule (50); it may also be
fluorescent by itself once linked. Once excess capture systems 30'
are removed, the fluorescent molecules are excited at the
appropriate wavelength and fluorescent intensity is read out. The
amount of luminescence or fluorescence detected is proportional to
the amount of target nucleic acid 40 in the sample 60.
[0154] In some sandwich assay embodiments, wherein the first
capture system 30 forms a triple helix with the target DNA, the
second capture system 30' can comprise a triple helix intercalator
or interactor. Some examples of triple helix intercalators that can
be used are bis-4-aminoquinolines and extended ethidium bromide
analogues. Examples of ethidium bromide analogues are given in
Wilson et al. Biochemistry, V. 32, pp. 10614-10621 (1993). The
embodiment where the first capture system 30 is a single stranded
nucleic acid which binds double stranded target molecules can
employ this type of second capture system 30'.
[0155] The second capture system 30' can be covalently bound to the
readable substrate or by way of a linker molecule 20', or just a
covalent bond. In other embodiments, the linker 20' between the
second capture system and luminescent substrate contains a specific
binding pair, which interacts non-covalently (e.g., biotin-avidin
complex). The linker must be long enough to allow for the
interactor to bind the double or triple stranded nucleic acid. When
the readable substrate is bound directly to the interactor, careful
consideration is paid to the binding site of the substrate on the
interactor, so as to preclude steric blocking of substrate
binding.
[0156] Unbound second capture systems 30' are removed in
wash/supernatant removal steps. Particles can be sequestered and
the unbound capture systems removed. Alternatively, manual or
automated pipetting steps are employed to wash the walls of
reaction vessels. The wash/supernatant removal step ensures
accurate quantification, e.g., by removing components that can
interfere with the readout reaction, by removing components that
contribute to background noise or that non-specifically bind to
reaction components.
[0157] Once the unbound labeled capture systems are washed away,
the second step of the reaction may occur either in the same vessel
or another one after automatic transfer by the machine.
[0158] In some embodiments, the readout reaction will use
luminescence. Depending on the luminescent reaction chosen, it will
be apparent to one of ordinary skill in the art how long to carry
out the reaction, and at what temperature, to ensure accurate and
reliable quantification, as parameters for carrying out luminescent
reactions are known. If conditions are not optimal, empirical
optimization can be carried out to determine reaction parameters.
The two capture systems may be added simultaneously. After
incubation, unbound material is washed away and detection may
occur.
[0159] In other embodiments, chemical or physical detection of the
label 50 is done. It will be apparent to one of ordinary skill in
the art how long to carry out the reaction, and at what
temperature, to ensure accurate and reliable quantification, as
parameters for carrying out detection are known. If conditions are
not optimal, empirical optimization can be carried out to determine
reaction parameters. The two capture systems (30 and 30') may be
added simultaneously. After incubation, unbound material is washed
away and detection may occur.
[0160] Competitive Method
[0161] In the competitive method, a known concentration of labeled
nucleic acid 40' is added to the reaction with the target nucleic
acid 40. The label 50'' is linked to the nucleic acid 40' via a
linker 20'', described supra. The labeled nucleic acid competes
with the unlabeled target nucleic acid for binding to the first
capture system 30. After an incubation time, the excess of labeled
nucleic acid fragments are removed in concert with an optional wash
step and the readout reaction is started, either in the same vessel
or another one after automatic transfer by the machine. Signal is
then measured and the intensity is inversely proportional to
nucleic acid concentration in the sample 60.
[0162] In some embodiments, readable detection is the detection of
a fluorescent substrate. The second capture system 30' is bound via
a linker 20' to a fluorescent dye or molecule (50). Once excess of
labeled nucleic acids 40' is removed, the fluorescent molecules are
excited at the appropriate wavelength and fluorescent intensity is
read out, either in the same vessel or another one after automatic
transfer by the machine. The amount of fluorescence detected is
inversely proportional to the amount of target in the sample
60.
[0163] In various competitive and sandwich assay embodiments, a
reagent which quenches a given luminescence reaction can be added
after luminescence detection in order to allow for subsequent wells
to be assayed without refractive or reflective cross-talk between
wells. Such reagents are disclosed in U.S. Pat. No. 5,744,320,
incorporated by reference in its entirety. These reagents can be
used for both sandwich and competitive assays.
[0164] The amount of target nucleic acid molecules present in the
sample is then compared to that a value that corresponds to the
amount of nucleic acids in sample from a subject that does not have
a particular disease. For example, if a sample analyzed from a
subject suspected of having cancer has an amount of target nucleic
acid molecules that is at least two fold greater, at least 4 fold
greater, at least 5 fold greater, or at least 10 to 100 fold
greater than that from a sample from a subject without canceris
indicative of a pathological condition. In embodiments, an amount
of cell free nucleic acid of at least about 5 ng/ml or at least
about 10 ng/ml is indicative of a pathological state.
[0165] Single Step Capture System Method
[0166] In some embodiments, first capture system 30 may contain a
nucleic acid interacting molecule or a set of molecules and a label
50. Once nucleic acid interaction occurred, label may react with
the sequestered conjugate and may become active. Once impurities
are removed by a washing step, the signal is then measured with the
appropriate method, either in the same vessel or another one after
automatic transfer by the machine. Intensity is proportional to
nucleic acid concentration in the sample 60.
[0167] In some embodiments, readable detection is the detection of
a fluorescent substrate. The capture system 30 is bound via a
linker 20 to a fluorescent dye or molecule (50). Once impurities
are removed by a washing step, the fluorescent molecules are
excited at the appropriate wavelength and fluorescent intensity is
read out, either in the same vessel or another one after automatic
transfer by the machine.
[0168] The capture system 30 is linked to the support 10 via a
linker 20, directly or indirectly. In some embodiments capture
system 30 is linked to the wall of the vessel, directly or
indirectly.
[0169] The label 50 is linked to the capture system 30 via a linker
20, directly or indirectly. It may react with the structure
resulting of interaction between capture system and nucleic acid,
or with a free part of the sequestered nucleic acid.
[0170] In certain embodiments, if the label 50 is linked to the
capture system 30 via a binding pair (e.g., biotin/streptavidin . .
. ) it may be added simultaneously with the sample or in a second
step after washing of the impurities. This further addition may
occur in the same vessel or another one after automatic transfer by
the machine. It is then possible to add a new washing step to
remove excess of unbound label.
[0171] In certain embodiments, the label 50 may be a molecule that
acquires power, or that becomes readable, only when reacting with
nucleic acid.
[0172] In some embodiments the capture system 30 may act as the
label 50. It may interact with the target nucleic acid 40 and then,
after acquiring power, be detected with an appropriate method.
[0173] The amount of target nucleic acid molecules present in the
sample is then compared to a value that corresponds to an amount of
nucleic acids in a sample from a subject that does not have a
particular disease. For example, if a sample analyzed from a
subject suspected of having cancer has a number of target nucleic
acid molecules that is at least two fold greater, at least 4 fold
greater, at least 5 fold greater, or at least 10 to 100 fold or
more greater than that from a sample from a subject without cancer
is indicative of a pathological state. In embodiments, an amount of
cell free nucleic acid of at least about 5 ng/ml or at least 10
ng/ml is indicative of a pathological state, i.e., a disease or
disorder.
[0174] Kits
[0175] Another aspect of the disclosure provides kits. In
embodiments, a kit for quantifying a target nucleic acid in a
sample comprises (i) a first nucleic acid interactor; (ii)
optionally, a second nucleic acid interactor; (iii) a label,
optionally bound to the first interactor; and (iv) instructions for
use. In embodiments, the kit further comprises control nucleic acid
fragments of different sizes and sequences.
[0176] In some embodiments, a kit comprises a first capture system
as described herein. In embodiments, the first capture system
comprises a first capture molecule, such as a nucleic acid
interactor bound to a support. In embodiments, the support and/or
the capture molecule are labeled with a readable label as described
herein.
[0177] In some embodiments, a kit further comprises a stock
solution of a control nucleic acid. The control nucleic acid can be
diluted and used to generate a standard curve to provide for
quantification of a target nucleic acid in a sample. In
embodiments, the control nucleic acid is a set of nucleic acids
each having a different size, for example from about 200 to about
1000 base pairs and any size in between.
[0178] In some embodiments, the kit further comprises instructions
for use. In embodiments, the instructions provide for
implementation of steps of the methods as described herein. A
determination of the result of the number or amount of target
nucleic acids in the sample is then compared to that of a control
sample and/or to a cutoff value as provided in the kit. In
embodiments, a kit may further comprise a control sample from a
subject not known to have a pathological condition. In embodiments,
a cutoff value indicative of a pathological state is at least about
5 ng/ml or 10 ng/ml of nucleic acid.
[0179] In embodiments, a kit further comprises a second capture
system as described herein. A second capture system comprises a
capture molecule, such as a nucleic acid interactor that is labeled
with a readable label. The second capture molecule may be the same
or different than the first capture molecule. In embodiments, both
capture molecules have similar spectral properties.
[0180] In some embodiments, a kit further comprises one or more
other nucleic acid interactor molecules.
[0181] In one embodiment, a kit further comprises a known quantity
of a labeled nucleic acid molecule. This known quantity of labeled
nucleic acid molecule is used to generate a standard curve and to
compete for binding with the unknown target nucleic acid in the
sample. In some embodiments, the known quantity of labeled nucleic
acid is an isolated double stranded DNA, isolated mRNA, isolated
single stranded DNA, isolated miRNA, and combinations thereof. When
multiple types of known nucleic acids are utilized, they each may
be labeled with a different readable label so that each may be
detected in a single sample.
[0182] The present invention is further illustrated by reference to
the Examples below. However, it should be noted that these
Examples, like the embodiments described above, are illustrative
and are not to be construed as restricting the enabled scope of the
invention in any way.
Example 1A
[0183] One example of a sandwich based assay uses second capture
systems 30' labeled with acridinium. The sample is added to the
reaction vessel containing a first capture system 30, which binds
to target nucleic acid 40. This capture system is linked, via a
linker 20, to a paramagnetic microparticle. The target nucleic acid
40 is incubated with the capture system 30 and the target nucleic
acid 40 binds to the capture system, forming a conjugate (e.g., an
intercalating complex). After an optional wash/supernatant removal
step, a second capture system 30' is added. This second system is
labeled with a chemiluminescent acridinium compound. It binds to
nucleic acid/first capture system conjugate. After incubation time,
a magnetic field is applied to the vessel and the paramagnetic
microparticles are sequestered to a wall of the reaction vessel.
The vessel is then washed to remove unbound material, i.e., excess
of the labeled second capture system. The light generation reaction
is started, after addition of a specific reagent (reagents
containing, e.g., hydrogen peroxide, acidic medium, and sodium
hydroxide) that chemically excites acridinium. The luminescence is
measured by an analyzer. The intensity of luminescence is
proportional to nucleic acid concentration in the crude sample. The
absolute quantity of nucleic acid can be determined by plotting the
results of the experiment on a standard curve generated with known
quantities of nucleic acids, taking into consideration sample loss
due to handling.
[0184] The above example can be further modified as follows: The
second capture system 30' is a biotin-labeled compound. It binds to
sequestered target nucleic acid 40. Avidin labeled with acridinium
is added to the reaction along with the second capture system 30'.
Avidin reacts with biotin linked to the intercalating complex.
After an incubation time, a magnetic field is applied to the
reaction vessel, which serves to sequester the microparticles to a
wall of the reaction vessel. The vessel is washed to remove unbound
material, i.e., excess of the second capture system 30' and avidin
labeled acridinium. The magnetic field is discontinued and the
luminescence reaction is started, after addition of a specific
reagent (e.g., hydrogen peroxide, acidic medium, and then sodium
hydroxide or Bayer reagent) that triggers a chemical excitation of
acridinium.
Example 1B
[0185] The sample 60 along with the first capture system 30 are
added to a reaction vessel, either manually or in an automated
fashion. The capture system is linked, via a linker 20, to a
paramagnetic microparticle. The linker 20 is labeled itself with a
cryptate molecule. The reaction mixture incubates and the nucleic
acid binds with the capture system, forming a conjugate.
Optionally, a wash step can be employed at this point to remove
impurities from the sample 60. To the previous mixture, the second
capture system 30' is added. This second system is labeled with a
XL665-molecule. It binds to nucleic acid/first capture system
conjugate. After an incubation time, a magnetic field is applied to
the vessel which attracts the microparticles to a wall of the
reaction vessel. The vessel is washed to remove unbound material,
i.e., excess of the XL665-labeled second capture system. The
magnetic field is discontinued and the light generation reaction is
started by the reading instrument, by light excitation of the
cryptate at 337 nm. Cryptate emits light which excites XL665, which
emits light (665 nm). The fluorescence intensity is then measured
and quantified by the instrument.
Example 1C
[0186] The reagents in Example 1A can be further modified to
include dioxetane phosphate-labeled- or ruthenium labeled-second
capture system 30'. After wash and supernatant removal steps, the
reaction of light generation is started, after manual or automated
addition of a specific Beckman reagent (when using
dioxentane--Lumi-Phos 530, phosphatase alcaline, surfactant
fluorescein) or a specific Roche reagent (ruthenium--TPA, oxidizes
ruthenium). The luminescence is then measured by the analyzer. The
linker 20 can comprise specific binding pair, such as avidin and
biotin, one of which will be attached to the microparticle.
Example 1D
Dade Behring Reagents
[0187] A sample 60 containing target nucleic acid 40 is added,
either manually or in an automated fashion, to a vessel containing
a first capture system 30, which binds to the target nucleic acid
40. This capture system is linked, via a linker 20, to a chemibead
microparticle (i.e., the support). The reaction mixture incubates
and the nucleic acid binds with the capture system, forming a
conjugate. Optionally at this point, a wash step can be employed to
remove any sample impurities. A second capture system 30' is then
added to the reaction vessel. This second system labeled with a
specific binding pair member, e.g., biotin. The second capture
system 30' binds to the sequestered nucleic acid, as described
supra. After an incubation time, a magnetic field is applied in
order to sequester the paramagnetic chemibead microparticles to a
wall of the reaction vessel. The vessel is washed to remove unbound
material, i.e., excess of the labeled second capture system. The
magnetic field is removed and label catchers linked to sensibead
microparticles are added. The label catcher is the other member of
the specific binding pair, e.g., avidin. It binds to the label 50
of the second capture system 30'. After an incubation time, a
magnetic field is applied to the vessel to sequester the
chemibead-sensibead complexes to a wall of the reaction vessel. The
vessel is washed to remove unbound material, i.e., excess of
unbound sensibead microparticles. The magnetic field is
discontinued and the light generation reaction is started, by
excitation (680 nm) of the chemibead microparticles. Chemibeads
emit light which excites sensibeads (FRET). The fluorescence
emitted by sensibead microparticles (520-620 nm) is then measured
and quantified by the analyzer.
[0188] In a similar implementation, as described above, the second
capture system 30' is directly linked to a sensibead microparticle,
instead of indirectly through a specific binding pair. The reaction
is carried out as described above, without the addition of the
additional reagent (because of the direct sensibead linkage to the
second capture system).
Example 2A
Acridinium Labeled Nucleic Acid for Competition Assay
[0189] Paramagnetic microparticles are used as the support, in
order to concentrate the target nucleic acid 40 and competing
nucleic acid 40'. Light is produced by direct excitation of
acridinium. Acridinium labeled nucleic acid fragments are added in
known concentration to the reaction vessel, with the sample 60
containing target nucleic acid 40. The reaction mixture incubates
with the capture system in the vessel, and the nucleic acid,
labeled or not, binds with a capture system, linked, via a linker
20, to a paramagnetic microparticle, forming a conjugate, e.g., an
intercalating complex. After a set incubation time, a magnetic
field is applied to the vessel, which attracts the paramagnetic
microparticles to a wall of the reaction vessel. The vessel is
washed to remove unbound material, i.e., excess of labeled nucleic
acid fragments 40' and other crude components of the sample 60. The
magnetic field is discontinued and the light generation reaction is
started, after addition and mixture (either manually or automated)
of a specific reagent (e.g., hydrogen peroxide, acidic medium, and
then sodium hydroxide or Bayer reagent) that chemically excites
acridinium. The luminescence is then measured by the detector of
the instrument.
Example 2B
Nucleic Acids Indirectly Labeled with Acridinium
[0190] Example 2A can be further modified by using competing
labeled nucleic acid 40' labeled with biotin or avidin, instead of
a direct label of acridinium. The competing nucleic acid 40' is
added to the reaction in concert with an avidin or biotin molecule
labeled with acridinium, either in an automated fashion or
manually. Avidin binds biotin (which is linked to the sequestered
competing nucleic acid conjugate). After an incubation time, a
magnetic field is applied to the vessel and sequesters the
paramagnetic microparticles to a wall of the reaction vessel. The
vessel is washed to remove unbound material, i.e., excess of
labeled nucleic acid fragments 40' and unbound luminescent
molecules, and other crude components of the sample 60. The
magnetic field is discontinued and the light generation reaction is
started, after manual or automated addition of a specific reagent
that chemically excites acridinium. The luminescence is then
measured by the instrument. The intensity of luminescence is
inversely proportional to the nucleic acid concentration in the
crude sample 60.
Example 2C
XL665 Label
[0191] The competing nucleic acid 40' exemplified in Example 2A can
be modified to include a XL665 label instead of acridinium. The
reaction is carried out in the same fashion as Example 2A, except
no additional reagent is added to generate the light emission
reaction. The first linker 20 is derivatized with a cryptate
molecule. Reaction of light generation is started by the analyzer,
by excitation of the cryptate at 337 nm. Cryptate and XL665 are a
FRET pair, so excitation of cryptate causes the excitation of
XL665. The XL665 fluorescence intensity is measured by the
analyzer.
Example 2D
[0192] The reagents in Example 2A can be further modified to
include dioxetane phosphate-labeled nucleic acid or ruthenium
labeled nucleic acid as the labeled competing nucleic acid 40'.
After wash and supernatant removal steps, the light generation
reaction is started by manual or automated addition of a specific
Beckman reagent (when using dioxentane--Lumi-Phos 530, alkaline
phosphatase, surfactant fluorescein) or a specific Roche reagent
(ruthenium-tris(2-pyridylmethyl)amine that serves to oxidize
ruthenium). The luminescence is then measured by the analyzer.
Example 2E
Dade Behring Reagents
[0193] Labeled nucleic acid fragments 40' in known concentrations
are added to a reaction vessel, along with the sample 60 containing
a target nucleic acid 40, and a first capture system 30. The
reaction mixture incubates and the nucleic acids, labeled or not,
bind with a capture system, linked, via a linker 20, to a chemibead
microparticle (i.e., the support 10). After an incubation time, a
magnetic field is applied and sequesters the paramagnetic chemibead
microparticles to a wall of the reaction vessel. The vessel is
washed to remove unbound material, i.e., excess of labeled nucleic
acid fragments 40'. Label catchers linked to sensibead
microparticles are added to the reaction. The label 50 and label
catchers can be specific binding pairs, for example, biotin and
avidin, respectively. The label catcher reacts with the label 50
linked to the nucleic acid conjugate. After an incubation time, a
magnetic field is applied to the vessel and attracts the
chemibead-sensibead complexes to a wall of the reaction vessel. The
vessel is washed to remove unbound material, i.e., excess of
sensibead microparticles. The light generation reaction is started,
by excitation (680 nm) of the chemibead microparticles. Chemibeads
emit light which in turn electronically excites sensibead
microparticles. The fluorescence emitted by sensibead
microparticles (520-620 nm) is measured by the analyzer.
Example 3
[0194] The present inventors have found that measurements in the
present method are more accurate if a set of two or more
interactors is used. Specifically, the inventors have found that
the amount of observed fluorescence or other signal used to
quantify the amount of the nucleic acid in a sample is dependent on
the length and (less so) on the sequence of target nucleic acid
present in a sample.
[0195] In an experiment employing DNA fragments of 432, about 500,
about 800 and 1400 bp, wherein a single interactor was used,
fluorescence was measured at 568 nm, 526 nm, 502 nm and both its
measured and its calculated total values varied considerably, with
longer fragments producing higher values that were four or more
times higher than those of the shortest fragment. The variation in
fluorescence persisted at different concentrations of fragment. As
expected, fluorescence was a linear function of concentration.
Fluorescence was also found to vary but less so according to the
sequence of the DNA fragment when the size was kept approximately
constant. For example in one experiment fragments of 203 bp having
disparate sequences produced calculated fluorescence as low as
9.156 and as high as 29.257.
[0196] Such differences especially those due to size will be
significantly reduced or altogether eliminated by use of dual
interactors.
[0197] Interactors, that are chemical molecules, may react
differently according to length or sequence of nucleic acids.
Particularly if interactors are intercalating agents, our
experience clearly exhibited such dependence.
[0198] We tested intercalating agents with several type of nucleic
acid sequences. In this experiment, we measured fluorescence
emitted by the DNA/intercalator complex.
[0199] We observed that the fluorescence signal was not constant if
we analysed the same concentration of five different sequences of
variable lengths (FIG. 4 A X1-X5). The intercalating agent used was
Picogreen. We repeated a similar experiment with another sequence
and another intercalating agent, Popo3. Results were drastically
different (FIG. 4, B X6-X10), indicating that the fluorescence
signal varies with the length of the detected DNA.
[0200] However, when we tested a unique sequence at different
concentrations, signal intensity is proportional to nucleic acid
concentration. This experience was repeated with several sequences
(X1 to X5) used in the previous experiment (FIG. 5).
TABLE-US-00001 TABLE 1 Sequences of X1-X5 X1-432 bp (SEQ ID NO: 1)
actcttctgg tccccacaga ctcagagaga acccaccatg gtgctgtctc ctgccgacaa
gaccaacgtc aaggccgcct ggggtaaggt cggcgcgcac gctggcgagt atggtgcgga
ggccctggag aggatgttcc tgtccttccc caccaccaag acctacttcc cgcacttcga
cctgagccac ggctctgccc aggttaaggg ccacggcaag aaggtggccg acgcgctgac
caacgccgtg gcgcacgtgg acgacatgcc caacgcgctg tccgccctga gcgacctgca
cgcgcacaag cttcgggtgg acccggtcaa cttcaagctc ctaagccact gcctgctggt
gaccctggcc gcccacctcc ccgccgagtt cacccctgcg gtgcacgcct ccctggacaa
gttcctggct tc X2-500 bp (SEQ ID NO: 2) atggcaagaa agtgctcggt
gcctttagtg atggcctggc tcacctggac aacctcaagg agtcctttgg ggatctgtcc
actcctgatg ctgttatggg caaccctaag gtgaaggctc tcaggctcct gggcaacgtg
ctggtctgtg tgctggccca tcactttggc aaagaattca acatttgctt ctgacacaac
tgtgttcact agcaacctca aacagacacc atggtgcatc tgactcctga ggagaagtct
gccgttactg ccctgtgggg caaggtgaac gtggatgaag ttggtggtga ggccctgggc
aggctgctgg tggtctaccc ttggacccag aggttctttg gcacctttgc cacactgagt
gagctgcact gtgacaagct gcacgtggat cctgagaact ccccaccagt gcaggctgcc
tatcagaaag tggtggctgg tgtggctaat gccctggccc acaagtatca ctaagctcgc
X3-800 bp (SEQ ID NO: 3) tatatgttta ttctttttat catctggaaa
cattcattat agttactgtc actaatcctt cacacttcca aaatctgaca agtttgacat
aggaaaaaaa tgggggaaat gtagatgaaa gagtttctat catctggaaa attgtgttat
aaaaattaag atgtctatcc cctctaggaa ttcctatgag ggatccttca cagtctaaag
gaaatgaagt ttgacaatgg tacaattcag ttttgaattt ttgctcttac tttctatatc
ctaacctttt ccagcttatt tcttgttaat tgttlltgga aagtatgcaa tgctctttta
agggaaaaaa aatcttctaa tgcacttatt taccttcatt tatatgtgtt tgtttctgaa
atgaagaatc aagctttggt cattccggaa gtcgtgtagc atgctccaat ttgaagtgac
tgagatcttt tgtgacttac agaggagaag gaaatgtttt aaaaattggc ttatgccagt
ctccttactc tgaaattctc aattttcttg tattacagga taaatataaa tatcatctca
ccaaattgta ccagcattgt cctaaacatt taaattttcc tttatltttg gcctttaaaa
ttagaaaatt ttctccagtt gctgcactta gctttttaat tttgttttct tttcacttac
cagactactg gtatgatctc tttcactgca gtatgattgc tgctatggtc aclittagag
gagattgtgg gaaggttagg atttggagga aacatgacaa aaaagggaag X4-1400 bp
(SEQ ID NO: 4) aagagagagc gcacacgcac acacccgccg cgcgcactcg
cgcacggacc cgcacgggga cagctcggaa gtcatcagtt ccatgggcga gatgctgctg
ctggcgagat gtctgctgct agtcctcgtc tcctcgctgc tggtatgctc gggactggcg
tgcggaccgg gcagggggtt cgggaagagg aggcacccca aaaagctgac ccctttagcc
tacaagcagt ttatccccaa tgtggccgag aagaccctag gcgccagcgg aaggtatgaa
gggaagatct ccagaaactc cgagcgattt aaggaactca cccccaatta caaccccgac
atcatattta aggatgaaga aaacaccgga gcggacaggc tgatgactca gaggtgtaag
gacaagttga acgctttggc catctcggtg atgaaccagt ggccaggagt gaaactgcgg
gtgaccgagg gctgggacga agatggccac cactcagagg agtctctgca ctacgagggc
cgcgcagtgg acatcaccac gtctgaccgc gaccgcagca agtacggcat gctggcccgc
ctggcggtgg aggccggctt cgactgggtg tactacgagt ccaaggcaca tatccactgc
tcggtgaaag cagagaactc ggtggcggcc aaatcgggag gctgcttccc gggctcggcc
acggtgcacc tggagcaggg cggcaccaag ctggtgaagg acctgagccc cggggaccgc
gtgctggcgg cggacgacca gggccggctg ctctacagcg acttcctcac tttcctggac
cgcgacgacg gcgccaagaa ggtcttctac gtgatcgaga cgcgggagcc gcgcgagcgc
ctgctgctca ccgccgcgca cctgctcttt gtggcgccgc acaacgactc ggccaccggg
gagcccgagg cgtcctcggg ctcggggccg ccttccgggg gcgcactggg gcctcgggcg
ctgttcgcca gccgcgtgcg cccgggccag cgcgtgtacg tggtggccga gcgtgacggg
gaccgccggc tcctgcccgc cgctgtgcac agcgtgaccc taagcgagga ggccgcgggc
gcctacgcgc cgctcacggc ccagggcacc attctcatca accgggtgct ggcctcgtgc
tacgcggtca tcgaggagca cagctgggcg caccgggcct tcgcgccctt ccgcctggcg
cacgcgctcc tggctgcact ggcgcccgcg cgcacggacc gcggcgggga cagcggcggc
ggggaccgcg ggggcggcgg cggcagagta gccctaaccg ctccaggtgc tgccgacgct
ccgggtgcgg gggccaccgc gggcatccac tggtactcgc X5-317 bp (SEQ ID NO:
5) ggtgagcggc ctccgaagcg gagcggggct ctgaggagac actttttttt
tcctccctcc ttccctcctc tcctcctccc ttcccttccc ctctcctccc ctctctcctc
cttcccccct cggtccgccg gagcctgctg gggcgagcgg ttggtattgc aggcgcttgc
tctccggggc cgcccggcgg gtagctggcg gggggaggag gcaggaaccg cgatggcgcc
tcagaagcac ggcggtgggg gagggggcgg ctcggggccc agcgcggggt ccgggggagg
cggcttcggg ggttcggcgg cggtggc
[0201] Thus, in light of these experiments, we can conclude that
the observed discrepancies are not due to a problem of
quantification, but rather to a problem of interaction between
intercalators and nucleic acid length and/or sequence. These
problems are corrected by use of more than one intercalator, such
as two, three four etc.
[0202] For that purpose, we tested five different sequences of
similar lengths, with either Picogreen or Popo3 (FIG. 6). We
observed a sequence dependence of signal intensity, regardless of
which the intercalating agent was used.
TABLE-US-00002 TABLE 2 Sequences of S1-S5 bp S1-653 bp (SEQ ID NO:
6) atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac
aacctcaagg agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag
gtgaaggctc tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc
aaagaattca acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc
atggtgcatc tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac
gtggatgaag ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag
aggttctttg gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat
cctgagaact ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat
gccctggccc acaagtatca ctaagctcgc tttcttgctg tccaatttct attaaaggtt
cctttgttcc ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgagcatct
ggattctgcc taataaaaaa catttatttt agtgctcggt gcctttagtg atggcctggc
cgg S2-627 bp (SEQ ID NO: 7) gcacctttgc cacactgagt gagctgcact
gtgacaagct gcacgtggat cctgagaact tcaggctcct gggcaacgtg ctggtctgtg
tgctggccca tcactttggc aaagaattca ccccaccagt gcaggctgcc tatcagaaag
tggtggctgg tgtggctaat gccctggccc acaagtatca ctaagctcgc tttcttgctg
tccaatttct attaaaggtt cctttgttcc acatttgctt ctgacacaac tgtgttcact
agcaacctca aacagacacc atggtgcatc tgactcctga ggagaagtct gccgttactg
ccctgtgggg caaggtgaac gtggatgaag ttggtggtga ggccctgggc aggctgctgg
tggtctaccc ttggacccag aggttctttg agtcctttgg ggatctgtcc actcctgatg
ctgttatggg caaccctaag gtgaaggctc atggcaagaa agtgctcggt gcctttagtg
atggcctggc tcacctggac aacctcaagg ctaagtccaa ctactaaact gggggatatt
atgaagggcc ttgagcatct ggattctgcc taataaaaaa catttalttt cattgct
S3-682 bp (SEQ ID NO: 8) acatttgctt ctgacacaac tgtgttcact
agcaacctca aacagacacc atggtgcatc tgactcctga ggagaagtct gccgttactg
ccctgtgggg caaggtgaac gtggatgaag ttggtggtga ggccctgggc aggctgctgg
tggtctaccc ttggacccag aggttctttg agtcctttgg ggatctgtcc actcctgatg
ctgttatggg caaccctaag gtgaaggctc atggcaagaa agtgctcggt gcctttagtg
atggcctggc tcacctggac aacctcaagg gcacctttgc cacactgagt gagctgcact
gtgacaagct gcacgtggat cctgagaact tcaggctcct gggcaacgtg ctggtctgtg
tgctggccca tcactttggc aaagaattca ccccaccagt gcaggctgcc tatcagaaag
tggtggctgg tgtggctaat gccctggccc acaagtatca ctaagctcgc tttcttgctg
tccaatttct attaaaggtt cctttgttcc ctaagtccaa ctactaaact gggggatatt
atgaagggcc ttgagcatct ggattctgcc taataaaaaa catttatttt acatttgctt
ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc gc S4-560 bp
(SEQ ID NO: 9) agtcctttgg ggatctgtcc actcctgatg ctgttatggg
caaccctaag gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc
tcacctggac aacctcaagg gcacctttgc cacactgagt gagctgcact gtgacaagct
gcacgtggat cctgagaact ttggtggtga ggccctgggc aggctgctgg tggtctaccc
ttggacccag aggttctttg tgactcctga ggagaagtct gccgttactg ccctgtgggg
caaggtgaac gtggatgaag acatttgctt ctgacacaac tgtgttcact agcaacctca
aacagacacc atggtgcatc tcaggctcct gggcaacgtg ctggtctgtg tgctggccca
tcactttggc aaagaattca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg
tgtggctaat gccctggccc acaagtatca ctaagctcgc tttcttgctg tccaatttct
attaaaggtt cctttgttcc ctaagtccaa ctactaaact S5-585 bp (SEQ ID NO:
10) ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag
aggttctttg tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac
gtggatgaag acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc
atggtgcatc agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag
gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac
aacctcaagg gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat
cctgagaact tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc
aaagaattca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat
gccctggccc acaagtatca ctaagctcgc tttcttgctg tccaatttct attaaaggtt
cctttgttcc ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgag
[0203] The reduction or elimination of length and sequence
dependence of the signal, is illustrated by the use of several
intercalating agents: picogreen, popo 3, yoyo 1, orange acridin, at
equimolar ratios. Results are shown in FIGS. 7 A (X6-X10) and
B(S1-S5). The use of a set of interactors, such as intercalating
agents, confers accuracy to the method.
TABLE-US-00003 TABLE 3 Sequences of X6-X10 X6-358 bp (SEQ ID NO:
11) atgaccctct ccggcggcgg cagcgccagc gacatgtccg gccagacggt
gctgacggcc gaggacgtgg acatcgatgt ggtgggcgag ggcgacgacg ggctggaaga
gaaggacagc gacgcaggtt gcgatagccc cgcggggccg ccggagctgc gcctggacga
ggcggacgag gtgcccccgg cggcacccca tcacggacag cctcagccgc cccaccagca
gcccctgaca ttgcccaagg aggcggccgg agccggggcc ggaccggggg gcgacgtggg
cgcgccggag gcggacggct gcaagggcgg tgttggcggc gaggagggcg gcgcgagcgg
cggcgggc >X7-64 0 bp (SEQ ID NO: 12) gtgcggggga agatgtagca
gcttcttctc cgaaccaacc ctttgccttc ggacttctcc ggggccagca gccgcccgac
caggggcccg gggccacggg ctcagccgac gaccatgggc tccgtgtcca accagcagtt
tgcaggtggc tgcgccaagg cggcagaaga ggcgcccgag gaggcgccgg aggacgcggc
ccgggcggcg gacgagcctc agctgctgca cggtgcgggc atctgtaagt ggttcaacgt
gcgcatgggg ttcggcttcc tgtccatgac cgcccgcgcc ggggtcgcgc tcgacccccc
agtggatgtc tttgtgcacc agagtaagct gcacatggaa gggttccgga gcttgaagga
gggtgaggca gtggagttca cctttaagaa gtcagccaag ggtctggaat ccatccgtgt
caccggacct ggtggagtat tctgtattgg gagtgagagg cggccaaaag gaaagagcat
gcagaagcgc agatcaaaag gagacaggtg ctacaactgt ggaggtctag atcatcatgc
caaggaatgc aagctgccac cccagcccaa gaagtgccac ttctgccaga gcatcagcca
tatggtagcc tcatgtccgc >X8-794 bp (SEQ ID NO: 13) attataaatc
tagagactcc aggattttaa cgttctgctg gactgagctg gttgcctcat gttattatgc
aggcaactca ctttatccca atttcttgat acttttcctt ctggaggtcc tatttctcta
acatcttcca gaaaagtctt aaagctgcct taaccttttt tccagtccac ctcttaaatt
ttttcctcct cttcctctat actaacatga gtgtggatcc agcttgtccc caaagcttgc
cttgctttga agcatccgac tgtaaagaat cttcacctat gcctgtgatt tgtgggcctg
aagaaaacta tccatccttg caaatgtctt ctgctgagat gcctcacacg gagactgtct
ctcctcttcc ttcctccatg gatctgctta ttcaggacag ccctgattct tccaccagtc
ccaaaggcaa acaacccact tctgcagaga agagtgtcgc aaaaaaggaa gacaaggtcc
cggtcaagaa acagaagacc agaactgtgt tctcttccac ccagctgtgt gtactcaatg
atagatttca gagacagaaa tacctcagcc tccagcagat gcaagaactc tccaacatcc
tgaacctcag ctacaaacag gtgaagacct ggttccagaa ccagagaatg aaatctaaga
ggtggcagaa aaacaactgg ccgaagaata gcaatggtgt gacgcagaag gcctcagcac
ctacctaccc cagcctttac tcttcctacc accagggatg cctggtgaac ccgactggga
accttccaat gtggagcaac cagacctgga acaattcaac ctggagt >X9-924 bp
(SEQ ID NO: 14) ctggggagag tatatactga atttagcttc tgagacatga
tgctcttcct ttttaattaa cccagaactt agcagcttat ctatttctct aatctcaaaa
catccttaaa ctgggggtga tacttgagtg agagaatttt gcaggtatta aatgaactat
cttctttttt ttttttctct gagacagagt cttgctctgt cacccaggct ggagtgcagt
ggcgtgatct cagctcactg caacctccgc ctcccgggtt caagtgattc tcctgcctca
gcctcctgag tagctgggat tacaggtgcg tgccaccgtg cccagctaat ttttgtgttt
ttagtagaga cggggtttca ccatgttggc catgctggtc ttgaactcct gacctcgtga
tctgcccacc tcggcctccc aaagtgctgg aattataggc gtgagccacc gcgcccagca
aagaacttct aaccttcata acctgacagg tgttctcgag gccagggtct ctctttctgt
cctttcacga tgctctgcat cccttggatg tgccagtttc tgggggaaga gtagtccttt
gttacatgca tgagtcagtg aacagggaat gggtgaatga catttgtggg taggttattt
ctagaagtta ggtgggcagc ttggaaggca gaggcacttc tacagactat tccctggggc
cacacgtagg ttcttgaatc ccgaatggaa aggggagatt gataactggt gtgtttatgt
tcttacaagt cttctgcctt ttaaaatcca gtcccaggac atcaaagctc tgcagaaaga
actcgagcaa tttgccaagc tcctgaagca gaagaggatc accctgggat atacacaggc
cgatgtgggg ctcaccctgg gggttctatt tgggaaggta ttca >X10-1300 bp
(SEQ ID NO: 15) atcagcccac caggagacct cgcccgccgc tcccccgggc
tccccggcca tgtctcccgc ccggctccgg ccccgactgc acttctgcct ggtcctgttg
ctgctgctgg tggtgccggc ggcatggggc tgcgggccgg gtcgggtggt gggcagccgc
cggcgaccgc cacgcaaact cgtgccgctc gcctacaagc agttcagccc caatgtgccc
gagaagaccc tgggcgccag cggacgctat gaaggcaaga tcgctcgcag ctccgagcgc
ttcaaggagc tcacccccaa ttacaatcca gacatcatct tcaaggacga ggagaacaca
ggcgccgacc gcctcatgac ccagcgctgc aaggaccgcc tgaactcgct ggctatctcg
gtgatgaacc agtggcccgg tgtgaagctg cgggtgaccg agggctggga cgaggacggc
caccactcag aggagtccct gcattatgag ggccgcgcgg tggacatcac cacatcagac
cgcgaccgca ataagtatgg actgctggcg cgcttggcag tggaggccgg ctttgactgg
gtgtattacg agtcaaaggc ccacgtgcat tgctccgtca agtccgagca ctcggccgca
gccaagacgg gcggctgctt ccctgccgga gcccaggtac gcctggagag tggggcgcgt
gtggccttgt cagccgtgag gccgggagac cgtgtgctgg ccatggggga ggatgggagc
cccaccttca gcgatgtgct cattttcctg gaccgcgagc ctcacaggct gagagccttc
caggtcatcg agactcagga ccccccacgc cgcctggcac tcacacccgc tcacctgctc
tctacggctg acaatcacac ggagccggca gcccgcttcc gggccacatt tgccagccac
gtgcagcctg gccagtacgt gctggtggct ggggtgccag gcctgcagcc tgcccgcgtg
gcagctgtct ctacacacgt ggccctcggg gcctacgccc cgctcacaaa gcatgggaca
ctggtggtgg aggatgtggt ggcaccctgc ttcgcggccg tggctgacca ccacctggct
cagttggcct tctggcccct gagactcttt cacagcttgg catggggcag ctggaccccg
ggggagggtg tgcattggta cccccagctg ctctaccgcc tggggcgtct cctgctagaa
gagggcagct tccacccact gggcatgtcc ggggcaggga gctgaaagga
ctccaccgct
Although the invention has been described in terms of exemplary
embodiments, it is not limited thereto. The claims should be
construed broadly to include other embodiments of the invention
that can be made by ones of ordinary skill in the art in light of
the present disclosure.
Sequence CWU 1
1
151432DNAHomo sapiens 1actcttctgg tccccacaga ctcagagaga acccaccatg
gtgctgtctc ctgccgacaa 60gaccaacgtc aaggccgcct ggggtaaggt cggcgcgcac
gctggcgagt atggtgcgga 120ggccctggag aggatgttcc tgtccttccc
caccaccaag acctacttcc cgcacttcga 180cctgagccac ggctctgccc
aggttaaggg ccacggcaag aaggtggccg acgcgctgac 240caacgccgtg
gcgcacgtgg acgacatgcc caacgcgctg tccgccctga gcgacctgca
300cgcgcacaag cttcgggtgg acccggtcaa cttcaagctc ctaagccact
gcctgctggt 360gaccctggcc gcccacctcc ccgccgagtt cacccctgcg
gtgcacgcct ccctggacaa 420gttcctggct tc 4322500DNAHomo sapiens
2atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac aacctcaagg
60agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag gtgaaggctc
120tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc
aaagaattca 180acatttgctt ctgacacaac tgtgttcact agcaacctca
aacagacacc atggtgcatc 240tgactcctga ggagaagtct gccgttactg
ccctgtgggg caaggtgaac gtggatgaag 300ttggtggtga ggccctgggc
aggctgctgg tggtctaccc ttggacccag aggttctttg 360gcacctttgc
cacactgagt gagctgcact gtgacaagct gcacgtggat cctgagaact
420ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat
gccctggccc 480acaagtatca ctaagctcgc 5003800DNAHomo sapiens
3tatatgttta ttctttttat catctggaaa cattcattat agttactgtc actaatcctt
60cacacttcca aaatctgaca agtttgacat aggaaaaaaa tgggggaaat gtagatgaaa
120gagtttctat cattttgaaa attgtgttat aaaaattaag atgtctatcc
cctctaggaa 180ttcctatgag ggatccttca cagtctaaag gaaatgaagt
ttgacaatgg tacaattcag 240ttttgaattt ttgctcttac tttctatatc
ctaacctttt ccagcttatt tcttgttaat 300tgtttttgga aagtatgcaa
tgctctttta agggaaaaaa aatcttctaa tgcacttatt 360taccttcatt
tatatgtgtt tgtttctgaa atgaagaatc aagctttggt cattccggaa
420gtcgtgtagc atgctccaat ttgaagtgac tgagatcttt tgtgacttac
agaggagaag 480gaaatgtttt aaaaattggc ttatgccagt ctccttactc
tgaaattctc aattttcttg 540tattacagga taaatataaa tatcatctca
ccaaattgta ccagcattgt cctaaacatt 600taaattttcc tttatttttg
gcctttaaaa ttagaaaatt ttctccagtt gctgcactta 660gctttttaat
tttgttttct tttcacttac cagactactg gtatgatctc tttcactgca
720gtatgattgc tgctatggtc acttttagag gagattgtgg gaaggttagg
atttggagga 780aacatgacaa aaaagggaag 80041400DNAHomo sapiens
4aagagagagc gcacacgcac acacccgccg cgcgcactcg cgcacggacc cgcacgggga
60cagctcggaa gtcatcagtt ccatgggcga gatgctgctg ctggcgagat gtctgctgct
120agtcctcgtc tcctcgctgc tggtatgctc gggactggcg tgcggaccgg
gcagggggtt 180cgggaagagg aggcacccca aaaagctgac ccctttagcc
tacaagcagt ttatccccaa 240tgtggccgag aagaccctag gcgccagcgg
aaggtatgaa gggaagatct ccagaaactc 300cgagcgattt aaggaactca
cccccaatta caaccccgac atcatattta aggatgaaga 360aaacaccgga
gcggacaggc tgatgactca gaggtgtaag gacaagttga acgctttggc
420catctcggtg atgaaccagt ggccaggagt gaaactgcgg gtgaccgagg
gctgggacga 480agatggccac cactcagagg agtctctgca ctacgagggc
cgcgcagtgg acatcaccac 540gtctgaccgc gaccgcagca agtacggcat
gctggcccgc ctggcggtgg aggccggctt 600cgactgggtg tactacgagt
ccaaggcaca tatccactgc tcggtgaaag cagagaactc 660ggtggcggcc
aaatcgggag gctgcttccc gggctcggcc acggtgcacc tggagcaggg
720cggcaccaag ctggtgaagg acctgagccc cggggaccgc gtgctggcgg
cggacgacca 780gggccggctg ctctacagcg acttcctcac tttcctggac
cgcgacgacg gcgccaagaa 840ggtcttctac gtgatcgaga cgcgggagcc
gcgcgagcgc ctgctgctca ccgccgcgca 900cctgctcttt gtggcgccgc
acaacgactc ggccaccggg gagcccgagg cgtcctcggg 960ctcggggccg
ccttccgggg gcgcactggg gcctcgggcg ctgttcgcca gccgcgtgcg
1020cccgggccag cgcgtgtacg tggtggccga gcgtgacggg gaccgccggc
tcctgcccgc 1080cgctgtgcac agcgtgaccc taagcgagga ggccgcgggc
gcctacgcgc cgctcacggc 1140ccagggcacc attctcatca accgggtgct
ggcctcgtgc tacgcggtca tcgaggagca 1200cagctgggcg caccgggcct
tcgcgccctt ccgcctggcg cacgcgctcc tggctgcact 1260ggcgcccgcg
cgcacggacc gcggcgggga cagcggcggc ggggaccgcg ggggcggcgg
1320cggcagagta gccctaaccg ctccaggtgc tgccgacgct ccgggtgcgg
gggccaccgc 1380gggcatccac tggtactcgc 14005317DNAHomo sapiens
5ggtgagcggc ctccgaagcg gagcggggct ctgaggagac actttttttt tcctccctcc
60ttccctcctc tcctcctccc ttcccttccc ctctcctccc ctctctcctc cttcccccct
120cggtccgccg gagcctgctg gggcgagcgg ttggtattgc aggcgcttgc
tctccggggc 180cgcccggcgg gtagctggcg gggggaggag gcaggaaccg
cgatggcgcc tcagaagcac 240ggcggtgggg gagggggcgg ctcggggccc
agcgcggggt ccgggggagg cggcttcggg 300ggttcggcgg cggtggc
3176653DNAHomo sapiens 6atggcaagaa agtgctcggt gcctttagtg atggcctggc
tcacctggac aacctcaagg 60agtcctttgg ggatctgtcc actcctgatg ctgttatggg
caaccctaag gtgaaggctc 120tcaggctcct gggcaacgtg ctggtctgtg
tgctggccca tcactttggc aaagaattca 180acatttgctt ctgacacaac
tgtgttcact agcaacctca aacagacacc atggtgcatc 240tgactcctga
ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag
300ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag
aggttctttg 360gcacctttgc cacactgagt gagctgcact gtgacaagct
gcacgtggat cctgagaact 420ccccaccagt gcaggctgcc tatcagaaag
tggtggctgg tgtggctaat gccctggccc 480acaagtatca ctaagctcgc
tttcttgctg tccaatttct attaaaggtt cctttgttcc 540ctaagtccaa
ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc
600taataaaaaa catttatttt agtgctcggt gcctttagtg atggcctggc cgg
6537627DNAHomo sapiens 7gcacctttgc cacactgagt gagctgcact gtgacaagct
gcacgtggat cctgagaact 60tcaggctcct gggcaacgtg ctggtctgtg tgctggccca
tcactttggc aaagaattca 120ccccaccagt gcaggctgcc tatcagaaag
tggtggctgg tgtggctaat gccctggccc 180acaagtatca ctaagctcgc
tttcttgctg tccaatttct attaaaggtt cctttgttcc 240acatttgctt
ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc
300tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac
gtggatgaag 360ttggtggtga ggccctgggc aggctgctgg tggtctaccc
ttggacccag aggttctttg 420agtcctttgg ggatctgtcc actcctgatg
ctgttatggg caaccctaag gtgaaggctc 480atggcaagaa agtgctcggt
gcctttagtg atggcctggc tcacctggac aacctcaagg 540ctaagtccaa
ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc
600taataaaaaa catttatttt cattgct 6278682DNAHomo sapiens 8acatttgctt
ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc 60tgactcctga
ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag
120ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag
aggttctttg 180agtcctttgg ggatctgtcc actcctgatg ctgttatggg
caaccctaag gtgaaggctc 240atggcaagaa agtgctcggt gcctttagtg
atggcctggc tcacctggac aacctcaagg 300gcacctttgc cacactgagt
gagctgcact gtgacaagct gcacgtggat cctgagaact 360tcaggctcct
gggcaacgtg ctggtctgtg tgctggccca tcactttggc aaagaattca
420ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat
gccctggccc 480acaagtatca ctaagctcgc tttcttgctg tccaatttct
attaaaggtt cctttgttcc 540ctaagtccaa ctactaaact gggggatatt
atgaagggcc ttgagcatct ggattctgcc 600taataaaaaa catttatttt
acatttgctt ctgacacaac tgtgttcact agcaacctca 660aacagacacc
atggtgcatc gc 6829558DNAHomo sapiens 9agtcctttgg ggatctgtcc
actcctgatg ctgttatggg caaccctaag gtgaaggctc 60atggcaagaa agtgctcggt
gcctttagtg atggcctggc tcacctggac aacctcaagg 120gcacctttgc
cacactgagt gagctgcact gtgacaagct gcacgtggat cctgagaact
180ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag
aggttctttg 240tgactcctga ggagaagtct gccgttactg ccctgtgggg
caaggtgaac gtggatgaag 300acatttgctt ctgacacaac tgtgttcact
agcaacctca aacagacacc atggtgcatc 360tcaggctcct gggcaacgtg
ctggtctgtg tgctggccca tcactttggc aaagaattca 420ccccaccagt
gcaggctgcc tatcagaaag tggtggctgg tgtggctaat gccctggccc
480acaagtatca ctaagctcgc tttcttgctg tccaatttct attaaaggtt
cctttgttcc 540ctaagtccaa ctactaaa 55810585DNAHomo sapiens
10ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag aggttctttg
60tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag
120acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc
atggtgcatc 180agtcctttgg ggatctgtcc actcctgatg ctgttatggg
caaccctaag gtgaaggctc 240atggcaagaa agtgctcggt gcctttagtg
atggcctggc tcacctggac aacctcaagg 300gcacctttgc cacactgagt
gagctgcact gtgacaagct gcacgtggat cctgagaact 360tcaggctcct
gggcaacgtg ctggtctgtg tgctggccca tcactttggc aaagaattca
420ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat
gccctggccc 480acaagtatca ctaagctcgc tttcttgctg tccaatttct
attaaaggtt cctttgttcc 540ctaagtccaa ctactaaact gggggatatt
atgaagggcc ttgag 58511358DNAHomo sapiens 11atgaccctct ccggcggcgg
cagcgccagc gacatgtccg gccagacggt gctgacggcc 60gaggacgtgg acatcgatgt
ggtgggcgag ggcgacgacg ggctggaaga gaaggacagc 120gacgcaggtt
gcgatagccc cgcggggccg ccggagctgc gcctggacga ggcggacgag
180gtgcccccgg cggcacccca tcacggacag cctcagccgc cccaccagca
gcccctgaca 240ttgcccaagg aggcggccgg agccggggcc ggaccggggg
gcgacgtggg cgcgccggag 300gcggacggct gcaagggcgg tgttggcggc
gaggagggcg gcgcgagcgg cggcgggc 35812640DNAHomo sapiens 12gtgcggggga
agatgtagca gcttcttctc cgaaccaacc ctttgccttc ggacttctcc 60ggggccagca
gccgcccgac caggggcccg gggccacggg ctcagccgac gaccatgggc
120tccgtgtcca accagcagtt tgcaggtggc tgcgccaagg cggcagaaga
ggcgcccgag 180gaggcgccgg aggacgcggc ccgggcggcg gacgagcctc
agctgctgca cggtgcgggc 240atctgtaagt ggttcaacgt gcgcatgggg
ttcggcttcc tgtccatgac cgcccgcgcc 300ggggtcgcgc tcgacccccc
agtggatgtc tttgtgcacc agagtaagct gcacatggaa 360gggttccgga
gcttgaagga gggtgaggca gtggagttca cctttaagaa gtcagccaag
420ggtctggaat ccatccgtgt caccggacct ggtggagtat tctgtattgg
gagtgagagg 480cggccaaaag gaaagagcat gcagaagcgc agatcaaaag
gagacaggtg ctacaactgt 540ggaggtctag atcatcatgc caaggaatgc
aagctgccac cccagcccaa gaagtgccac 600ttctgccaga gcatcagcca
tatggtagcc tcatgtccgc 64013836DNAHomo sapiens 13attataaatc
tagagactcc aggattttaa cgttctgctg gactgagctg gttgcctcat 60gttattatgc
aggcaactca ctttatccca atttcttgat acttttcctt ctggaggtcc
120tatttctcta acatcttcca gaaaagtctt aaagctgcct taaccttttt
tccagtccac 180ctcttaaatt ttttcctcct cttcctctat actaacatga
gtgtggatcc agcttgtccc 240caaagcttgc cttgctttga agcatccgac
tgtaaagaat cttcacctat gcctgtgatt 300tgtgggcctg aagaaaacta
tccatccttg caaatgtctt ctgctgagat gcctcacacg 360gagactgtct
ctcctcttcc ttcctccatg gatctgctta ttcaggacag ccctgattct
420tccaccagtc ccaaaggcaa acaacccact tctgcagaga agagtgtcgc
aaaaaaggaa 480gacaaggtcc cggtcaagaa acagaagacc agaactgtgt
tctcttccac ccagctgtgt 540gtactcaatg atagatttca gagacagaaa
tacctcagcc tccagcagat gcaagaactc 600tccaacatcc tgaacctcag
ctacaaacag gtgaagacct ggttccagaa ccagagaatg 660aaatctaaga
ggtggcagaa aaacaactgg ccgaagaata gcaatggtgt gacgcagaag
720gcctcagcac ctacctaccc cagcctttac tcttcctacc accagggatg
cctggtgaac 780ccgactggga accttccaat gtggagcaac cagacctgga
acaattcaac ctggag 83614924DNAHomo sapiens 14ctggggagag tatatactga
atttagcttc tgagacatga tgctcttcct ttttaattaa 60cccagaactt agcagcttat
ctatttctct aatctcaaaa catccttaaa ctgggggtga 120tacttgagtg
agagaatttt gcaggtatta aatgaactat cttctttttt ttttttcttt
180gagacagagt cttgctctgt cacccaggct ggagtgcagt ggcgtgatct
cagctcactg 240caacctccgc ctcccgggtt caagtgattc tcctgcctca
gcctcctgag tagctgggat 300tacaggtgcg tgccaccgtg cccagctaat
ttttgtgttt ttagtagaga cggggtttca 360ccatgttggc catgctggtc
ttgaactcct gacctcgtga tctgcccacc tcggcctccc 420aaagtgctgg
aattataggc gtgagccacc gcgcccagca aagaacttct aaccttcata
480acctgacagg tgttctcgag gccagggtct ctctttctgt cctttcacga
tgctctgcat 540cccttggatg tgccagtttc tgggggaaga gtagtccttt
gttacatgca tgagtcagtg 600aacagggaat gggtgaatga catttgtggg
taggttattt ctagaagtta ggtgggcagc 660ttggaaggca gaggcacttc
tacagactat tccttggggc cacacgtagg ttcttgaatc 720ccgaatggaa
aggggagatt gataactggt gtgtttatgt tcttacaagt cttctgcctt
780ttaaaatcca gtcccaggac atcaaagctc tgcagaaaga actcgagcaa
tttgccaagc 840tcctgaagca gaagaggatc accctgggat atacacaggc
cgatgtgggg ctcaccctgg 900gggttctatt tgggaaggta ttca
924151300DNAHomo sapiens 15atcagcccac caggagacct cgcccgccgc
tcccccgggc tccccggcca tgtctcccgc 60ccggctccgg ccccgactgc acttctgcct
ggtcctgttg ctgctgctgg tggtgccggc 120ggcatggggc tgcgggccgg
gtcgggtggt gggcagccgc cggcgaccgc cacgcaaact 180cgtgccgctc
gcctacaagc agttcagccc caatgtgccc gagaagaccc tgggcgccag
240cggacgctat gaaggcaaga tcgctcgcag ctccgagcgc ttcaaggagc
tcacccccaa 300ttacaatcca gacatcatct tcaaggacga ggagaacaca
ggcgccgacc gcctcatgac 360ccagcgctgc aaggaccgcc tgaactcgct
ggctatctcg gtgatgaacc agtggcccgg 420tgtgaagctg cgggtgaccg
agggctggga cgaggacggc caccactcag aggagtccct 480gcattatgag
ggccgcgcgg tggacatcac cacatcagac cgcgaccgca ataagtatgg
540actgctggcg cgcttggcag tggaggccgg ctttgactgg gtgtattacg
agtcaaaggc 600ccacgtgcat tgctccgtca agtccgagca ctcggccgca
gccaagacgg gcggctgctt 660ccctgccgga gcccaggtac gcctggagag
tggggcgcgt gtggccttgt cagccgtgag 720gccgggagac cgtgtgctgg
ccatggggga ggatgggagc cccaccttca gcgatgtgct 780cattttcctg
gaccgcgagc ctcacaggct gagagccttc caggtcatcg agactcagga
840ccccccacgc cgcctggcac tcacacccgc tcacctgctc tttacggctg
acaatcacac 900ggagccggca gcccgcttcc gggccacatt tgccagccac
gtgcagcctg gccagtacgt 960gctggtggct ggggtgccag gcctgcagcc
tgcccgcgtg gcagctgtct ctacacacgt 1020ggccctcggg gcctacgccc
cgctcacaaa gcatgggaca ctggtggtgg aggatgtggt 1080ggcatcctgc
ttcgcggccg tggctgacca ccacctggct cagttggcct tctggcccct
1140gagactcttt cacagcttgg catggggcag ctggaccccg ggggagggtg
tgcattggta 1200cccccagctg ctctaccgcc tggggcgtct cctgctagaa
gagggcagct tccacccact 1260gggcatgtcc ggggcaggga gctgaaagga
ctccaccgct 1300
* * * * *